Talk:MitoPedia: Fluorometry: Difference between revisions
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{{MitoPedia | |||
|description='''Fluorometry''' (or [[fluorimetry]]) is the general term given to the method of measuring the fluorescent emission of a substance following excitation by light at a shorter wavelength. | |||
}} | |||
__TOC__ | __TOC__ | ||
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== '''O2k-Fluo Smart-Module''' == | == '''O2k-Fluo Smart-Module''' == | ||
=== | === [[O2k-FluoRespirometer]] === | ||
[[ | |||
=== Select the Smart Fluo-Sensors === | === Select the Smart Fluo-Sensors === | ||
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:::: Connect the Smart Fluo-Sensor cable to the Fluo plug of the O2k, insert the male plug of the cable into the female Fluo plug. The red dot on the male plug faces straight upwards. Each Smart Fluo-Sensor can be used on O2k-Chamber A or B. The blue frame of the chamber window and the Smart Fluo-Sensor are specially designed to align in a specific rotational position with the cable extending horizontally to left (chamber A) and right (chamber B). The Smart Fluo-Sensor is carefully inserted into the window frame and rotated into final position, leaving no gap between window frame and sensor body. | :::: Connect the Smart Fluo-Sensor cable to the Fluo plug of the O2k, insert the male plug of the cable into the female Fluo plug. The red dot on the male plug faces straight upwards. Each Smart Fluo-Sensor can be used on O2k-Chamber A or B. The blue frame of the chamber window and the Smart Fluo-Sensor are specially designed to align in a specific rotational position with the cable extending horizontally to left (chamber A) and right (chamber B). The Smart Fluo-Sensor is carefully inserted into the window frame and rotated into final position, leaving no gap between window frame and sensor body. | ||
:::: To remove the Smart Fluo-Sensor, carefully pull out the sensor body with slight back and forth rotations. Do not pull the cable. | :::: To remove the Smart Fluo-Sensor, carefully pull out the sensor body with slight back and forth rotations. Do not pull the cable. | ||
=== Settings in [[DatLab | DatLab 7.4]] === | |||
:::*''-See:'' In the [[O2k control]] window in the [[Amperometric,Amp]] tab, set fluorescence intensity and amplification of the signal. | |||
== '''O2k-Fluo LED2-Module''' == | == '''O2k-Fluo LED2-Module''' == | ||
[[MiPNet17.05 O2k-Fluo LED2-Module]] | [[MiPNet17.05 O2k-Fluo LED2-Module]] | ||
=== Select the Fluorescence-Sensors === | === Select the Fluorescence-Sensors === | ||
:::: Switching between different excitation wavelengths and filters is achieved by simply exchanging the | :::: Switching between different excitation wavelengths and filters is achieved by simply exchanging the Fluo-Sensors. Two types of optical sensors are supplied with different LEDs for fluorescence excitation, and the effective spectra of the LEDs are modified by filters. | ||
:::: Select the Fluo-Sensor and filter set from section 6 of [[MiPNet17.05_O2k-Fluorescence_LED2-Module#Fluorophores|application-specific settings]]. Each Fluo-Sensor is delivered with a mounted filter set. | :::: Select the Fluo-Sensor and filter set from section 6 of [[MiPNet17.05_O2k-Fluorescence_LED2-Module#Fluorophores|application-specific settings]]. Each Fluo-Sensor is delivered with a mounted filter set. | ||
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=== Setup and connect O2k-Fluorescence LED2-Module === | === Setup and connect O2k-Fluorescence LED2-Module === | ||
[[File:Fluorescence-Control Unit lettered.jpg|400px|right|link=]] | [[File:Fluorescence-Control Unit lettered.jpg|400px|right|link=]] | ||
::::* Switch off the O2k with the power switch on the rear of the [[O2k-Main Unit]]. | ::::* Switch off the O2k with the power switch on the rear of the [[O2k-Main Unit]]. | ||
::::* Press the power switch on the front panel of the Fluorescence-Control Unit. Check that the two red/green control lights (O2k-Fluorescence LED2-Module B and upwards) or the central green control light are on. | |||
::::* Remove both blue [[O2k-Window Frame]]s. Insert the [[O2k-Window Tool]] around the outer rim of the window frame and unscrew in a counter clockwise direction. | ::::* Remove both blue [[O2k-Window Frame]]s. Insert the [[O2k-Window Tool]] around the outer rim of the window frame and unscrew in a counter clockwise direction. | ||
::::* Remove the '''Sensor-Guide''' (‘nose’) from the '''O2k-Front Fixation''' of the Fluorescence-Control Unit. | ::::* Remove the '''Sensor-Guide''' (‘nose’) from the '''O2k-Front Fixation''' of the Fluorescence-Control Unit. | ||
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:::: In this configuration the O2k can be used for [[high-resolution respirometry]] and fluorometry. It is not necessary to dismount the Fluorescence-Control Unit for basic HRR when a fluorescence signal is not recorded. | :::: In this configuration the O2k can be used for [[high-resolution respirometry]] and fluorometry. It is not necessary to dismount the Fluorescence-Control Unit for basic HRR when a fluorescence signal is not recorded. | ||
=== Settings in DatLab | |||
=== Settings in DatLab: Series E-G === | |||
[[File:Configuration.PNG|right|300px]] | [[File:Configuration.PNG|right|300px]] | ||
::::* From the DatLab menu choose [Oxygraph]/[O2k-Control]. | ::::* From the DatLab menu choose [Oxygraph]/[O2k-Control]. | ||
:::: In the '''O2k configuration | :::: In the '''[[O2k configuration]]''' window include serial number (#) of the sensors used. | ||
:::: In the '''O2k | :::: In the '''[[O2k control]]''' window in the ´Amperometric, Amp´ tab the Fluo/LED intensity and the gain (amplification) can be set. | ||
:::: '''Setting the LED Intensity from DatLab: O2k-Fluorescence LED2-Module Series B and higher (modules <u>without</u> mechanical selector switch)''' | :::: '''Setting the LED Intensity from DatLab: O2k-Fluorescence LED2-Module Series B and higher (modules <u>without</u> mechanical selector switch)''' | ||
::::* Set the desired light intensity (0 to 2000 mv) in the field "Amp Polarization Voltage [mV]". | ::::* Set the desired light intensity (0 to 2000 mv) in the field "Amp Polarization Voltage [mV]". | ||
::::* Click on "Send to Oxygraph" or "Connect to Oxygraph-2k" to apply the new settings. | ::::* Click on "Send to Oxygraph" or "Connect to Oxygraph-2k" to apply the new settings. | ||
::::* If any current >= 0 is set and the Fluorescence Module is switched on, the indicator light below the respective chamber on the Fluorescence-Control Unit will be green. If the current is 0 (the LED is not used) but the Fluorescence-Control_Unit is switched on, the indicator light will be red. | ::::* If any current >= 0 is set and the Fluorescence Module is switched on, the indicator light below the respective chamber on the Fluorescence-Control Unit will be green. If the current is 0 (the LED is not used) but the Fluorescence-Control_Unit is switched on, the indicator light will be red. | ||
:::: Limitations: For O2k-Series D the LED intensity must be set to the same value for both chambers. | :::: Limitations: For O2k-Series D the LED intensity must be set to the same value for both chambers. | ||
Note: The actual current (in mA) used to drive the LED is the value set in DatLab (Amp polarization voltage) divided by 100. | Note: The actual current (in mA) used to drive the LED is the value set in DatLab (Amp polarization voltage) divided by 100. | ||
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|- | |- | ||
|} | |} | ||
== '''General''' == | == '''General''' == | ||
=== Fluorescence dyes === | === Fluorescence dyes === | ||
{| class="wikitable" | {| class="wikitable" | ||
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! Application !!Sensor!! Filter set !! O2k output !! Light intensity (polarization voltage) - ''Note a'' !! Gain !! Comment | ! Application !!Sensor!! Filter set !! O2k output !! Light intensity (polarization voltage) - ''Note a'' !! Gain !! Comment | ||
|- | |- | ||
| [[Amplex UltraRed]]||[[Fluorescence-Sensor Green]] or Smart Fluo-Sensor Green || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type B]] || 100 - 500 || 1000 (at light intensity = 100) || | | [[Amplex UltraRed]]||[[Fluorescence-Sensor Green]] or Smart Fluo-Sensor Green || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type B]] || 100 - 500 || 1000 (at light intensity = 100) || at c(AmR) = 10 µM | ||
|- | |- | ||
| [[TMRM]]||[[Fluorescence-Sensor Green]]or Smart Fluo-Sensor Green || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type C]] || 200 - 500|| 1000 || at c(TMRM) = 2 µM | | [[TMRM]]||[[Fluorescence-Sensor Green]]or Smart Fluo-Sensor Green || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type C]] || 200 - 500|| 1000 || at c(TMRM) = 2 µM | ||
|- | |- | ||
| [[Safranin]] ||[[Fluorescence-Sensor Blue]]or Smart Fluo-Sensor Blue || [[Filter Set Saf|Saf]] || [[O2k_signal_and_output| Type C]] || 100 - | | [[Safranin]] ||[[Fluorescence-Sensor Blue]]or Smart Fluo-Sensor Blue || [[Filter Set Saf|Saf]] || [[O2k_signal_and_output| Type C]] || 100 - 500 || 1000 || at c(Saf) = 2 µM, | ||
|- | |- | ||
| [[Magnesium green]]||[[Fluorescence-Sensor Blue]]or Smart Fluo-Sensor Blue|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output| Type B]] || 100 - | | [[Magnesium green]]||[[Fluorescence-Sensor Blue]]or Smart Fluo-Sensor Blue|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output| Type B]] || 100 - 500|| 1000 || at c(Mg Green) = 2 µM | ||
|- | |- | ||
| [[Calcium green]]||[[Fluorescence-Sensor Blue]]or Smart Fluo-Sensor Blue|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output|Type A and C]] ||100 -300|| 1000 ||at c(Ca Green) = 2 µM | | [[Calcium green]]||[[Fluorescence-Sensor Blue]]or Smart Fluo-Sensor Blue|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output|Type A and C]] ||100 -300|| 1000 ||at c(Ca Green) = 2 µM | ||
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[[Image:Filter Set AmR.JPG|180px|right]] | [[Image:Filter Set AmR.JPG|180px|right]] | ||
::::* The set of filters for the determination of H<sub>2</sub>O<sub>2</sub> production with [[Amplex UltraRed]] should be used together with [[Fluorescence-Sensor Green | ::::* The set of filters for the determination of H<sub>2</sub>O<sub>2</sub> production with [[Amplex UltraRed]] should be used together with [[Fluorescence-Sensor Green]]. | ||
::::[[File:AmR excitation scan 1mA.png|300px]] | ::::[[File:AmR excitation scan 1mA.png|300px]] | ||
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==== Filter Set Saf ==== | ==== Filter Set Saf ==== | ||
[[Image:Filter_Set_Saf.JPG|180px|right]] | [[Image:Filter_Set_Saf.JPG|180px|right]] | ||
::::* Set of filters for the (qualitative) determination of mitochondrial membrane potential with [[Safranin]] should be used together with [[Fluorescence-Sensor Blue]] or [[Smart Fluo-Sensor Blue | ::::* Set of filters for the (qualitative) determination of mitochondrial membrane potential with [[Safranin]] should be used together with [[Fluorescence-Sensor Blue]] or [[Smart Fluo-Sensor Blue]]. | ||
::::[[File:Saf excitation scan 1mA.png|300px]] | ::::[[File:Saf excitation scan 1mA.png|300px]] | ||
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[[Image:Filter_Set_MgG_CaG.JPG|180px|right]] | [[Image:Filter_Set_MgG_CaG.JPG|180px|right]] | ||
::::* The set of filters for the determination of concentrations of Mg<sup>2+</sup> or Ca<sup>2+</sup> with the fluorophores [[Magnesium green]] and [[Calcium green]], respectively, should be used together with [[Fluorescence-Sensor Blue]] or [[Smart Fluo-Sensor Blue | ::::* The set of filters for the determination of concentrations of Mg<sup>2+</sup> or Ca<sup>2+</sup> with the fluorophores [[Magnesium green]] and [[Calcium green]], respectively, should be used together with [[Fluorescence-Sensor Blue]] or [[Smart Fluo-Sensor Blue]]. | ||
::::[[File:MgG excitation scan 1mA.png|300px]] | ::::[[File:MgG excitation scan 1mA.png|300px]] | ||
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::::The light intensity of the LED ([[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]) and the signal amplification ([[Fluorescence-Control_Unit#Gain|Gain]]) can be adjusted in a wide range. The table suggests initial values, which can be optimised for specific applications. | ::::The light intensity of the LED ([[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]) and the signal amplification ([[Fluorescence-Control_Unit#Gain|Gain]]) can be adjusted in a wide range. The table suggests initial values, which can be optimised for specific applications. | ||
::::* The settings depend on the concentration of the fluorophore, which vary between different applications. Therefore, only recommendations for specific fluorophore concentrations are given. In the Amplex UltraRed assay the fluorophore is formed during the experiment. | ::::* The settings depend on the concentration of the fluorophore, which vary between different applications. Therefore, only recommendations for specific fluorophore concentrations are given. In the Amplex UltraRed assay the fluorophore is formed during the experiment. | ||
::::* The recommendations apply to experiments at 37 °C. The | ::::* The recommendations apply to experiments at 37 °C. The fluorescence intensity increases strongly at lower temperatures. Then the light intensity is reduced to avoid off-scale signals. | ||
::::* The light intensity of the LEDs is set by the current control, independent for each fluorescence sensor (O2k-Chamber A and B). The current is controlled by DatLab in a very wide range for optimization according to sample and fluorophore requirements. At higher LED-intensity the optical sensitivity is increased, i.e. the signal change per concentration change is enhanced. However, even moderately intensive light may exert negative effects: (i) Damage to the sample reducing the biological activity. (ii) Damage to fluorophores catalyzing degradation and various side reactions. Therefore, the LED-intensity should be kept as low as compatible with a smooth signal, i.e. when the resolution is just not limited by noise or disturbances. The values indicated in the table [[O2k-Fluo_LED2-Module#Application-specific_settings|application specific settings]] are only suggestions to start with. It is recommended to optimize the light intensity specifically for each application. | |||
=== Gain, amplification === | === Gain, amplification === | ||
:::: The gain for the Amp channel can be set in the Control Table of the Oxygraph Control window, the section “Amp” to 1, 10, 100, or 1000. The gain setting will influence the Amp raw signal recorded in Volt. See [[O2k-Fluorescence_LED2-Module#Observing_the_fluorescence_signal| Observing the Fluorescence Signal]]. The amplified signal can be recorded in the range -10 to +10 V. | :::: The gain for the Amp channel can be set in the Control Table of the Oxygraph Control window, the section “Amp” to 1, 10, 100, or 1000. The gain setting will influence the Amp raw signal recorded in Volt. See [[O2k-Fluorescence_LED2-Module#Observing_the_fluorescence_signal| Observing the Fluorescence Signal]]. The amplified signal can be recorded in the range -10 to +10 V. | ||
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::::* [[Titration-Injection microPump |TiP2k]]: Our tests indicated that the fluorometric signal is not affected by the TIP2k needle inserted into the O2k-Chamber. | ::::* [[Titration-Injection microPump |TiP2k]]: Our tests indicated that the fluorometric signal is not affected by the TIP2k needle inserted into the O2k-Chamber. | ||
== '''Troubleshooting''' == | |||
=== Defective Fluo-Sensor or O2k or O2k-LED2 Fluo-Module=== | === Defective Fluo-Sensor or O2k or O2k-LED2 Fluo-Module=== | ||
::: If the raw fluorescence signal is 0 V with and | ::: If the raw fluorescence signal is 0 V with and without fluorescence dyes, it might be possible that your Fluo-Sensor is defective. How can you identify the problem? | ||
:::: Please go through the following check-list: | |||
:::: 1. Check that the [[Illumination on/off|illumination]] is | :::: 1. Check that the [[Illumination on/off|illumination]] is switched off in the ´[[O2k control]]´window. | ||
:::: 2. Check the settings for the Amperometric channel in the ´[[O2k control]]´ window, in the Amperometric,Amp tab. Gain for Fluo-Sensor and the Fluo intensity cannot be 0. | :::: 2. Check the settings for the [[Amperometric,Amp]]channel in the ´[[O2k control]]´ window, in the [[Amperometric,Amp]] tab. Gain for Fluo-Sensor and the Fluo intensity cannot be 0. | ||
:::: 3. The Fluo-Sensors are connected to the O2k and inserted into the O2k-Chamber in the right position (see above in the 1.3 Connect Smart Fluo-Sensor to O2k section). | :::: 3. The Fluo-Sensors are connected to the O2k and inserted into the O2k-Chamber in the right position (see above in the 1.3 Connect Smart Fluo-Sensor to O2k section). | ||
:::: 4. If all the above-mentioned steps are checked and the detected signal with your Fluo-Sensor is 0 | :::: 4. If all the above-mentioned steps are checked and the detected signal with your Fluo-Sensor is 0 (negative or very high) in order to localise the problem, perform the following test. Add distilled water or respiration medium into the O2k-Chamber, insert the black stopper and change the settings for fluorometric measurements (see above): | ||
::::::* 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B | ::::::* 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B | ||
::::::* 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A | ::::::* 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A | ||
:::::: If your Fluo-Sensor A is defective, the fluorescence signal should be 0 V both in chamber A and B. In this case, the Fluo-Sensor should be shipped to us in order to repair it. | :::::: If your Fluo-Sensor A is defective, the fluorescence signal should be 0 V with Fluo-Sensor A both in chamber A and B. In this case, the Fluo-Sensor should be shipped to us in order to repair it. | ||
:::::: If the O2k or the O2k-LED2 | :::::: If the O2k or the O2k-LED2 Fluo-Module are defective, 0 V or negative fluorescence signal can be observed just in one chamber with two different Fluo-Sensors (see example below). In this case, the O2k or the Fluorescence- Control Unit (Series D-G) should be shipped to us in order to repair it. | ||
::::: Please contact our [https://wiki.oroboros.at/index.php/O2k-Open_Support| customer support] to analyse your problem before shippment. Please always send us original DatLab files with the above-mentioned tests to evaluate the defective Fluo-Sensor performance. | ::::: Please contact our [https://wiki.oroboros.at/index.php/O2k-Open_Support| customer support] to analyse your problem before shippment. Please always send us original DatLab files with the above-mentioned tests to evaluate the defective Fluo-Sensor performance. | ||
:::: '''Problem: Defective O2k or O2k-LED2 Fluo-Module''' | :::: '''Problem: Defective O2k or O2k-LED2 Fluo-Module''' | ||
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::::[[File:Hassan Hazirah 2.test.png|700px]] | ::::[[File:Hassan Hazirah 2.test.png|700px]] | ||
::::[[File:Hassan Hazirah biological sample.png|700px]] | ::::[[File:Hassan Hazirah biological sample.png|700px]] | ||
:::: Analysing these tests after changing the Fluo-Sensors between the O2k-Chambers, it is obvious that the problem is related to the O2k or O2k-LED2 | :::: Analysing these tests after changing the Fluo-Sensors between the O2k-Chambers, it is obvious that the problem is related to the O2k or O2k-LED2 Fluo-Module, since the fluorescence signal is negative and unstable in chamber B with both Fluo-Sensors. In the third figure the fluorescence signal is very unstable and it makes measurement unpossible with biological sample. Both the O2k and the LED2 Fluo-Module should be shipped to our electronic workshop. | ||
=== The raw fluorescence signal [V] appears constantly at 10 V === | |||
::: If you see a constant raw signal at 10 V when using the [[O2k-Fluo Smart-Module]] or [[MiPNet17.05 O2k-Fluo LED2-Module | O2k-Fluo LED2-Module]], this is probably due to high light exposure to the photodiode. Check the following parameters: | |||
::::* The chamber illumination must be switched off: Check it in the [[O2k control]] window. | |||
::::* The photodiode filters must be inserted, otherwise the light emitted from the LED will not be filtered. Click [[Filter-Cap#Mounting_a_Filter-Cap|here]] more information on how to mount the filter cap. | |||
::::* Ensure that the [[O2k-Fluo_Smart-Module#Connect_Smart_Fluo-Sensor_to_O2k|Smart Fluo-Sensor is correctly inserted]] at the O2k frame of the chamber window. | |||
::::* Use [[O2k-Fluo_Smart-Module#Stoppers|black PEEK stoppers]]. | |||
=== Negative fluorescence signal=== | === Negative fluorescence signal=== | ||
::::Problem: The fluorescence | ::::Problem: The fluorescence signal of the Fluo-Sensor in chamber B is negative, while the other Fluo-Sensor has ~0 V. The same result occurs when the [https://bioblast.at/index.php/MiPNet17.05_O2k-Fluo_LED2-Module| O2k-LED2 Fluo-Module] is disconnected from the the O2k/switched off. | ||
::::Provided by Anne Laure Charles, FR_Strasbourg_Zoll J,anne laure charles <[email protected] | ::::Provided by Anne Laure Charles, FR_Strasbourg_Zoll J,anne laure charles <[email protected] | ||
::::[[File:Anne Laure Charles files.png|700px]] | ::::[[File:Anne Laure Charles files.png|700px]] | ||
:::: Answer: Connacting our electronic workshop, the problem might be related to wet plug of the | :::: Answer: Connacting our electronic workshop, the problem might be related to a wet plug of the Fluo-Sensors. The solution is to dry it out somehow ''e.g.'' hair dryer and let it run connected to the O2k for a couple of days in dry and warm environment. If these treatments cannot solve the problem and you get still negative fluorescence values, Fluo-Sensor should be sent back to us and our electronic partner will look at it and replace it if needed. | ||
[[Image:BB-Bioblast.jpg|left|40px|link=http://www.bioblast.at/index.php/Bioblast:About|Bioblast wiki]] | [[Image:BB-Bioblast.jpg|left|40px|link=http://www.bioblast.at/index.php/Bioblast:About|Bioblast wiki]] | ||
[[Image:O2k-Publications.jpg|left|116px|link=O2k-Publications: Topics|O2k-Publications in the MiPMap]] | [[Image:O2k-Publications.jpg|left|116px|link=O2k-Publications: Topics|O2k-Publications in the MiPMap]] | ||
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}} | }} | ||
</div> | </div> | ||
== Useful links: == | |||
::::» [[MiPNet22.11 O2k-FluoRespirometer manual| O2k-FluoRespirometer manual]] | |||
::::» [[O2k-Demo_experiments |O2k-Demo experiments]] | |||
::::» [[MiPNet06.05 Specifications | Sole source information]] | |||
::::» [[Oroboros O2k]] | |||
[[Image:SmartFluo.png|thumb|200px|right| Smart Fluo-Sensors]] | |||
[[File:Smart_Fluo-Sensor_-_connect_.png|thumb|100px|right|'''Orientation''' for connection]] | |||
[[File:Smart_Fluo-Sensor_RIGHT.jpg|thumb|100px|right|'''OK''', fully inserted]] | |||
[[File:Smart_Fluo-Sensor_WRONG.jpg|thumb|100px|right|'''WRONG''', not fully inserted]] | |||
[[File:Smart_Fluo-Sensor_inserted.jpg|thumb|100px|right|'''Horizontal''' cable routing]] | |||
=== O2k-Fluorometry Workshops === | |||
::::* [[Oroboros_Events#Next_O2k-Workshops|Oroboros O2k-Workshops]] | |||
== Popular Bioblast page == | == Popular Bioblast page == |
Latest revision as of 10:15, 26 May 2020
Description
Fluorometry (or fluorimetry) is the general term given to the method of measuring the fluorescent emission of a substance following excitation by light at a shorter wavelength.
» MitoPedia methods: Fluorometry
»
MitoPedia O2k and high-resolution respirometry:
O2k hardware
MitoPedia O2k and high-resolution respirometry:
O2k-Open Support
O2k-Fluo Smart-Module
O2k-FluoRespirometer
Select the Smart Fluo-Sensors
- Switching between different excitation wavelengths and filters is achieved by simply exchanging the Smart Fluo-Sensors. Two types of optical sensors are supplied with different LEDs for fluorescence excitation, and the effective spectra of the LEDs are modified by filters.
- Select the Fluo-Sensor and filter set from section 6 of application-specific settings. Each Fluo-Sensor is delivered with a mounted filter set.
Smart Fluo-Sensor Green
- Excitation LED 525 nm (dominant wavelength) with short pass filter, emission (red) with long pass filter, individual sensors are calibrated with sensor-specific memory and direct input into DatLab 7 to obtain reproducible light intensities with different sensors, including photodiode, Filter-Cap equipped with Filter Set AmR for Amplex UltraRed and TMRM measurements when delivered.
- Two units of this item are standard components of the O2k-FluoRespirometer and the O2k-Fluo Smart-Module. Smart Fluo-Sensors can be used with O2k-Series H and higher.
Smart Fluo-Sensor Blue
- Excitation LED 465 nm (dominant wavelenght) with short pass filter, emission (red) with long pass filter, individual sensors are calibrated with sensor-specific memory and direct input into DatLab 7 to obtain reproducible light intensities with different sensors, including photodiode, Filter-Cap equipped with Filter Set Saf for measurement of mitochondrial membrane potential with safranin and rhodamine 123. Filter Set MgG / CaG for Magnesium green and Calcium green measurements are included.
- Two units of this item are standard components of the O2k-FluoRespirometer and the O2k-Fluo Smart-Module. Smart Fluo-Sensors can be used with O2k-Series H and higher.
Setup and connect Smart Fluo-Sensor to O2k
- Connect the Smart Fluo-Sensor cable to the Fluo plug of the O2k, insert the male plug of the cable into the female Fluo plug. The red dot on the male plug faces straight upwards. Each Smart Fluo-Sensor can be used on O2k-Chamber A or B. The blue frame of the chamber window and the Smart Fluo-Sensor are specially designed to align in a specific rotational position with the cable extending horizontally to left (chamber A) and right (chamber B). The Smart Fluo-Sensor is carefully inserted into the window frame and rotated into final position, leaving no gap between window frame and sensor body.
- To remove the Smart Fluo-Sensor, carefully pull out the sensor body with slight back and forth rotations. Do not pull the cable.
Settings in DatLab 7.4
- -See: In the O2k control window in the Amperometric,Amp tab, set fluorescence intensity and amplification of the signal.
O2k-Fluo LED2-Module
MiPNet17.05 O2k-Fluo LED2-Module
Select the Fluorescence-Sensors
- Switching between different excitation wavelengths and filters is achieved by simply exchanging the Fluo-Sensors. Two types of optical sensors are supplied with different LEDs for fluorescence excitation, and the effective spectra of the LEDs are modified by filters.
- Select the Fluo-Sensor and filter set from section 6 of application-specific settings. Each Fluo-Sensor is delivered with a mounted filter set.
Fluorescence-Sensor Green
- Excitation LED 525 nm (dominant wavelength), photodiode, Filter-Cap equipped with Filter Set AmR for Amplex UltraRed measurements when delivered.
- Two units of this item are standard components of the O2k-Fluo LED2-Module.
Fluorescence-Sensor Blue
- Excitation LED 465 nm (dominant wavelength), photodiode, Filter-Cap equipped with Filter Set Saf for measurement of mitochondrial membrane potential with Safranin when delivered. Filter sets for Magnesium green® / Calcium green® measurements are included.
- Two units of this item are standard components of the O2k-Fluo LED2-Module.
Setup and connect O2k-Fluorescence LED2-Module
- Switch off the O2k with the power switch on the rear of the O2k-Main Unit.
- Press the power switch on the front panel of the Fluorescence-Control Unit. Check that the two red/green control lights (O2k-Fluorescence LED2-Module B and upwards) or the central green control light are on.
- Remove both blue O2k-Window Frames. Insert the O2k-Window Tool around the outer rim of the window frame and unscrew in a counter clockwise direction.
- Remove the Sensor-Guide (‘nose’) from the O2k-Front Fixation of the Fluorescence-Control Unit.
- Place the Fluorescence-Control Unit below the O2k-Chamber Block. Align the windows of the O2k-Front-Fixation with the windows of the O2k-Chamber Block, re-insert the O2k-Window Frames, and screw them finger-tight onto the O2k-Main Unit.
- Re-attach the Sensor-Guide to the O2k-Front-Fixation. (There is no need to use the fixation screw supplied with early models).
- Place the power-cables from the rear of the Fluorescence-Control Unit in the middle below the O2k-Main Unit from front to rear. Unplug the mains power cable of the O2k and plug it into the female plug of the Fluorescence-Control Unit. Insert the male plug of the Fluorescence-Control Unit into the mains socket at the rear of the O2k.
- Connect the amperometric cables attached to the side of the Fluorescence-Control Unit to the ‘Amp’ plugs (labeled "NO" in Series D-E) on the O2k-Main Unit.
- In this configuration the O2k can be used for high-resolution respirometry and fluorometry. It is not necessary to dismount the Fluorescence-Control Unit for basic HRR when a fluorescence signal is not recorded.
Settings in DatLab: Series E-G
- From the DatLab menu choose [Oxygraph]/[O2k-Control].
- In the O2k configuration window include serial number (#) of the sensors used.
- In the O2k control window in the ´Amperometric, Amp´ tab the Fluo/LED intensity and the gain (amplification) can be set.
- Setting the LED Intensity from DatLab: O2k-Fluorescence LED2-Module Series B and higher (modules without mechanical selector switch)
- Set the desired light intensity (0 to 2000 mv) in the field "Amp Polarization Voltage [mV]".
- Click on "Send to Oxygraph" or "Connect to Oxygraph-2k" to apply the new settings.
- If any current >= 0 is set and the Fluorescence Module is switched on, the indicator light below the respective chamber on the Fluorescence-Control Unit will be green. If the current is 0 (the LED is not used) but the Fluorescence-Control_Unit is switched on, the indicator light will be red.
- Limitations: For O2k-Series D the LED intensity must be set to the same value for both chambers.
- Setting the LED Intensity from DatLab: O2k-Fluorescence LED2-Module Series B and higher (modules without mechanical selector switch)
Note: The actual current (in mA) used to drive the LED is the value set in DatLab (Amp polarization voltage) divided by 100. Previously we quoted the suggested current. To simplify the operation we now state directly the required DatLab settings.
- Setting the LED Intensity from DatLab: O2k-Fluorescence LED2-Module Series A (modules with mechanical selector switch)
- O2k-Series A of the O2k-Fluorescence LED2-Module has a mechanical selector switch for each chamber on the front side of the Fluorescence-Control_Unit
- Set the switch located on the front panel of the Fluorescence-Control_Unit to position 9 with help of the small screw driver included with the Module.
- Follow the procedure described above for Series B and higher.
- For LED2-Module Series A it is still possible to set the LED current directly at the Fluorescence-Control_Unit. However, such set function is no longer in use. The relevant instructions can be found on the discussion page in the archive.
- Conversion of the LED intensities between the Fluorescence-Sensor of the O2k-Fluo LED2-Module (O2k-Series D to G) and the Smart Fluo-Sensor of the O2k-FluoRespirometer (O2k-Series H)
- The O2k-FluoRespirometer (O2k-Series H) and software DatLab 7, the fluorescent intensity (excitation) of the Smart Fluo-Sensors is set in 'Fluo intensity' units. The Smart Fluo-Sensors are pre-calibrated to guarantee similar fluorescence intensity among Smart Fluo-Sensors of the same colour, storing the sensor-specific 'gain value' in an internal memory. Technically speaking, DatLab 7 (i) reads this 'gain value' from a connected Smart Fluo-Sensor to adjust the output intensity and (ii) automatically corrects for the non-linear relationship between intensity and 'mV' to output the correct fluorescent intensity. In contrast, the fluorescent intensity (excitation) of the Fluorescence-Sensors of the O2k-Fluo LED2-Module (O2k-Series D to G) is set directly in 'mV' units. To set the same intensity for O2k-Fluo LED2-Module (O2k-Series D to G) Fluorescence-Sensor as for a O2k-FluoRespirometer (O2k-Series H) Smart Fluo-Sensor, we have created an Excel sheet that allows to calculate it.
Download the EXCEL calculation-sheet to adjust fluoarescence intensity |
General
Fluorescence dyes
Application | Sensor | Filter set | O2k output | Light intensity (polarization voltage) - Note a | Gain | Comment |
---|---|---|---|---|---|---|
Amplex UltraRed | Fluorescence-Sensor Green or Smart Fluo-Sensor Green | AmR | Type B | 100 - 500 | 1000 (at light intensity = 100) | at c(AmR) = 10 µM |
TMRM | Fluorescence-Sensor Greenor Smart Fluo-Sensor Green | AmR | Type C | 200 - 500 | 1000 | at c(TMRM) = 2 µM |
Safranin | Fluorescence-Sensor Blueor Smart Fluo-Sensor Blue | Saf | Type C | 100 - 500 | 1000 | at c(Saf) = 2 µM, |
Magnesium green | Fluorescence-Sensor Blueor Smart Fluo-Sensor Blue | MgG / CaG | Type B | 100 - 500 | 1000 | at c(Mg Green) = 2 µM |
Calcium green | Fluorescence-Sensor Blueor Smart Fluo-Sensor Blue | MgG / CaG | Type A and C | 100 -300 | 1000 | at c(Ca Green) = 2 µM |
Rhodamine 123 | Fluorescence-Sensor Blueor Smart Fluo-Sensor Blue | Filter Set Saf | Type III | 100 -300 | 1000 | at c(Rh123) = 1 µM |
Stoppers
- Use only black PEEK stoppers in conjunction with fluorometric measurements. The black stoppers can be used for all HRR applications. See MiPNet22.11 O2k-FluoRespirometer manual for calibration of the O2k-chamber volume, which is identical for PEEK and PVDF stoppers.
Selecting a Filter Set
- The Filter-Cap of each sensor can be removed for application of various filter combinations on the same optical sensor.
- Selection of a Filter Set: >> Application specific settings.
- The Filter-Cap of each sensor can be removed for application of various filter combinations on the same optical sensor.
Filter Set AmR
- The set of filters for the determination of H2O2 production with Amplex UltraRed should be used together with Fluorescence-Sensor Green.
- Excitation spectrum obtained with the Fluorescence-Sensor Green at 1 mA current supply and equipped with the Filter Set AmR.
- In the O2k-Fluo LED2-Module dye based-absorption filters in form of polymer films are used. If required, cut-off values related to the filters (short of a full spectrum) can be used:
- AmR excitation filter (SG 370): dye based short pass filter, 50% T: 535 nm, suppressing (<5% Transmission) lambda > 595 nm
- AmR emission filter (RL2001): dye based long pass filter, 50% T: 620 nm, suppressing (<2% transmission) lambda < 590 nm
- The spectra of excitation (LED filter: R370) and emission (photodiode filter: R2001) can be found on the webpage of our supplier, ROSCO: https://emea.rosco.com/de/node/1706 (please select the filter numbers to download the specific data sheets).
Filter Set Saf
- Set of filters for the (qualitative) determination of mitochondrial membrane potential with Safranin should be used together with Fluorescence-Sensor Blue or Smart Fluo-Sensor Blue.
- Excitation spectrum obtained with the Fluorescence-Sensor Blue at 1mA current supply and equipped with the Filter Set Saf.
- In the O2k-Fluo LED2-Module dye based absorption filters in form of polymer films are used. If required, cut-off values related to the filters (short of a full spectrum) can be used:
- Saf excitation filter: (SG357): dye based short pass filter, 50% T: 470 nm, suppressing (<5% Transmission) lambda > 530 nm
- Saf emission filter: (SG19): dye based long pass filter, 50% T: 600 nm, suppressing (<1% transmission) lambda < 550 nm
- The spectra of excitation (LED filter: R357) and emission (photodiode filter: R19) can be found on the webpage of our supplier, ROSCO: https://emea.rosco.com/de/node/1706 (please select the filter numbers to download the specific data sheets).
Filter Set MgG/CaG
- The set of filters for the determination of concentrations of Mg2+ or Ca2+ with the fluorophores Magnesium green and Calcium green, respectively, should be used together with Fluorescence-Sensor Blue or Smart Fluo-Sensor Blue.
- Excitation spectrum obtained with the Fluorescence-Sensor Blue at 1 mA current supply and equipped with the filter set for Magnesium green® / Calcium green®.
- In the O2k-Fluo LED2-Module dye based absorption filters in form of polymer films are used. If required, cut-off values related to the filters (short of a full spectrum) can be used:
- MgG/CaG excitation filter (SG59):dye based short pass filter, 50% T: not applicable (max. transmission < 50%), suppressing (<5% Transmission) lambda > 490 nm
- MgG/CaG emission filter (RL21):dye based long pass filter,50% T: 570 nm, suppressing (<1% transmission) lambda < 525 nm
- The spectra of excitation (LED filter: R59) and emission (photodiode filter: R21) can be found on the webpage of our supplier, ROSCO: https://emea.rosco.com/de/node/1706 (please select the filter numbers to download the specific data sheets).
Mounting a Filter-Cap
- Applies to: O2k-Fluo LED2-Module (O2k-Series D to G) Fluorescence Sensor and O2k-FluoRespirometer (O2k-Series H) with Smart Fluo-Sensors
- Dismounting: Pull the Filter-Cap straight from the sensor. The Filter-Cap Guide prevents rotational movements.
- Remove all filters and store them in the filter box labeled for this Filter Set.
- Insert the filters form the selected Filter Set: The round filters of each Filter Set fit to the round window of the Filter-Cap and cover the LED, the rectangular filters fit into the rectangular window of the Filter-Cap and cover the photodiode.
- Mounting: Hold sensor and filter cap in a vertical position above you. Align the Filter-Cap with the Filter-Cap Guide (small steel rod) protruding from the sensor. Press the Filter-Cap onto the sensor without rotational movements.
Control of LED-intensity
- The light intensity of the LED (LED-intensity) and the signal amplification (Gain) can be adjusted in a wide range. The table suggests initial values, which can be optimised for specific applications.
- The settings depend on the concentration of the fluorophore, which vary between different applications. Therefore, only recommendations for specific fluorophore concentrations are given. In the Amplex UltraRed assay the fluorophore is formed during the experiment.
- The recommendations apply to experiments at 37 °C. The fluorescence intensity increases strongly at lower temperatures. Then the light intensity is reduced to avoid off-scale signals.
- The light intensity of the LED (LED-intensity) and the signal amplification (Gain) can be adjusted in a wide range. The table suggests initial values, which can be optimised for specific applications.
- The light intensity of the LEDs is set by the current control, independent for each fluorescence sensor (O2k-Chamber A and B). The current is controlled by DatLab in a very wide range for optimization according to sample and fluorophore requirements. At higher LED-intensity the optical sensitivity is increased, i.e. the signal change per concentration change is enhanced. However, even moderately intensive light may exert negative effects: (i) Damage to the sample reducing the biological activity. (ii) Damage to fluorophores catalyzing degradation and various side reactions. Therefore, the LED-intensity should be kept as low as compatible with a smooth signal, i.e. when the resolution is just not limited by noise or disturbances. The values indicated in the table application specific settings are only suggestions to start with. It is recommended to optimize the light intensity specifically for each application.
Gain, amplification
- The gain for the Amp channel can be set in the Control Table of the Oxygraph Control window, the section “Amp” to 1, 10, 100, or 1000. The gain setting will influence the Amp raw signal recorded in Volt. See Observing the Fluorescence Signal. The amplified signal can be recorded in the range -10 to +10 V.
- The gain setting should be chosen to obtain a maximum signal well below 10 V. If the maximum observed raw signal was 9 V in an initial experiment, then the gain should be reduced to avoid "off scale" (>9.99 V). On the other hand, if the maximum recorded raw signal was considerable lower than 1 V (e.g. 0.2 V), the gain can be increased to avoid limitation of resolution by digital noise.
- Note for advanced users: At gain 1, a current of 1 nA is recorded as a voltage of 1 mV (0.001 V). At gain 100, 1 nA corresponds to 100 mV (0.1 V), as can be seen form the table below:
1
|
0.001 | 1000
|
+-10000
|
333
|
10
|
0.01 | 100
|
+-1000
|
33
|
1000
|
1 | 1
|
+-10
|
0.3
|
The fluorescence signal
- O2k signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [Amp]) as the primary signal.
- O2k output: type A, B, or C, or combinations.
Graph layout: Three plots are available in DatLab based on the recorded signal: Amp Raw Signal, Amp Calibrated, and Amp Slope. These plots can be selected from the drop-down lines and displayed with their check boxes either on the Y1 or Y2 [Graph layout / Select Plots].
- Amp Raw Signal displays the raw voltage (including amplification) as recorded by the O2k at a given gain setting.
- Amp Calibrated is the signal after calibration with the parameters set in the O2k-MultiSensor Calibration window.
- Amp slope is the time derivative of the calibrated signal, multiplied by 1000, in units [mV(conc. Unit during calibration)/s], so if the signal was calibrated in µM [nmol/ml] the unit of the slope is pmol/(s ml). To obtain the slope of the raw signal check the appropriate box in the calibration window (DatLab 5.1.0.130 and above).
- Graphs can be generated to display oxygen and fluorescence data, or several graphs can be added to display oxygen and fluorescence data separately. Layout templates are provided, which can be modified and saved as appropriate. All graph settings can be saved as user-defined layouts, see MiPNet19.01C DatLab Guide.
O2k-Fluorometry and the TIP2k
- TiP2k: Our tests indicated that the fluorometric signal is not affected by the TIP2k needle inserted into the O2k-Chamber.
Troubleshooting
Defective Fluo-Sensor or O2k or O2k-LED2 Fluo-Module
- If the raw fluorescence signal is 0 V with and without fluorescence dyes, it might be possible that your Fluo-Sensor is defective. How can you identify the problem?
- Please go through the following check-list:
- 1. Check that the illumination is switched off in the ´O2k control´window.
- If the raw fluorescence signal is 0 V with and without fluorescence dyes, it might be possible that your Fluo-Sensor is defective. How can you identify the problem?
- 2. Check the settings for the Amperometric,Ampchannel in the ´O2k control´ window, in the Amperometric,Amp tab. Gain for Fluo-Sensor and the Fluo intensity cannot be 0.
- 3. The Fluo-Sensors are connected to the O2k and inserted into the O2k-Chamber in the right position (see above in the 1.3 Connect Smart Fluo-Sensor to O2k section).
- 4. If all the above-mentioned steps are checked and the detected signal with your Fluo-Sensor is 0 (negative or very high) in order to localise the problem, perform the following test. Add distilled water or respiration medium into the O2k-Chamber, insert the black stopper and change the settings for fluorometric measurements (see above):
- 1. run: Fluo-Sensor A in chamber A and Fluo-Sensor B in chamber B
- 2. run: Fluo-Sensor A in chamber B and Fluo-Sensor B in chamber A
- If your Fluo-Sensor A is defective, the fluorescence signal should be 0 V with Fluo-Sensor A both in chamber A and B. In this case, the Fluo-Sensor should be shipped to us in order to repair it.
- If the O2k or the O2k-LED2 Fluo-Module are defective, 0 V or negative fluorescence signal can be observed just in one chamber with two different Fluo-Sensors (see example below). In this case, the O2k or the Fluorescence- Control Unit (Series D-G) should be shipped to us in order to repair it.
- Please contact our customer support to analyse your problem before shippment. Please always send us original DatLab files with the above-mentioned tests to evaluate the defective Fluo-Sensor performance.
- Problem: Defective O2k or O2k-LED2 Fluo-Module
- Provided by Hazirah Hassan, University Kebangsaan Malaysia, [email protected]
- Analysing these tests after changing the Fluo-Sensors between the O2k-Chambers, it is obvious that the problem is related to the O2k or O2k-LED2 Fluo-Module, since the fluorescence signal is negative and unstable in chamber B with both Fluo-Sensors. In the third figure the fluorescence signal is very unstable and it makes measurement unpossible with biological sample. Both the O2k and the LED2 Fluo-Module should be shipped to our electronic workshop.
- 4. If all the above-mentioned steps are checked and the detected signal with your Fluo-Sensor is 0 (negative or very high) in order to localise the problem, perform the following test. Add distilled water or respiration medium into the O2k-Chamber, insert the black stopper and change the settings for fluorometric measurements (see above):
The raw fluorescence signal [V] appears constantly at 10 V
- If you see a constant raw signal at 10 V when using the O2k-Fluo Smart-Module or O2k-Fluo LED2-Module, this is probably due to high light exposure to the photodiode. Check the following parameters:
- The chamber illumination must be switched off: Check it in the O2k control window.
- The photodiode filters must be inserted, otherwise the light emitted from the LED will not be filtered. Click here more information on how to mount the filter cap.
- Ensure that the Smart Fluo-Sensor is correctly inserted at the O2k frame of the chamber window.
- Use black PEEK stoppers.
- If you see a constant raw signal at 10 V when using the O2k-Fluo Smart-Module or O2k-Fluo LED2-Module, this is probably due to high light exposure to the photodiode. Check the following parameters:
Negative fluorescence signal
- Problem: The fluorescence signal of the Fluo-Sensor in chamber B is negative, while the other Fluo-Sensor has ~0 V. The same result occurs when the O2k-LED2 Fluo-Module is disconnected from the the O2k/switched off.
- Provided by Anne Laure Charles, FR_Strasbourg_Zoll J,anne laure charles <[email protected]
- Answer: Connacting our electronic workshop, the problem might be related to a wet plug of the Fluo-Sensors. The solution is to dry it out somehow e.g. hair dryer and let it run connected to the O2k for a couple of days in dry and warm environment. If these treatments cannot solve the problem and you get still negative fluorescence values, Fluo-Sensor should be sent back to us and our electronic partner will look at it and replace it if needed.
- » References
Sort in ascending/descending order by a click on one of the small symbols in squares below. Default sorting: chronological. Empty fields appear first in ascending order.
Was published in year | Has title | Organism | Tissue;cell | |
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MiPNet18.10 O2k-Specifications | 2024-03-26 | O2k-specifications for respirometry and comprehensive OXPHOS analysis. | Human | Fibroblast |
Lhuissier 2024 iScience | 2024 | Lhuissier C, Desquiret-Dumas V, Girona A, Alban J, Faure J, Cassereau J, Codron P, Lenaers G, Baris OR, Gueguen N, Chevrollier A (2024) Mitochondrial F0F1-ATP synthase governs the induction of mitochondrial fission. iScience 27:109808. https://doi.org/10.1016/j.isci.2024.109808 | Mouse | Fibroblast |
Balmaceda 2024 Biochim Biophys Acta Mol Basis Dis | 2024 | Balmaceda V, Komlodi T, Szibor M, Gnaiger E, Moore AL, Fernandez-Vizarra E, Viscomi C (2024) The striking differences in the bioenergetics of brain and liver mitochondria are enhanced in mitochondrial disease. Biochim Biophys Acta Mol Basis Dis 1870:167033. https://doi.org/10.1016/j.bbadis.2024.167033 | Mouse | Nervous system Liver |
Ravasz 2024 Sci Rep | 2024 | Ravasz D, Bui D, Nazarian S, Pallag G, Karnok N, Roberts J, Marzullo BP, Tennant DA, Greenwood B, Kitayev A, Hill C, Komlódi T, Doerrier C, Cunatova K, Fernandez-Vizarra E, Gnaiger E, Kiebish Michael A, Raska A, Kolev K, Czumbel B, Narain NR, Seyfried TN, Chinopoulos C (2024) Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia. Sci Rep 14:1729. https://doi.org/10.1038/s41598-024-51365-4 | Mouse | Heart Liver |
Fitzgerald 2024 J Cachexia Sarcopenia Muscle | 2024 | Fitzgerald LF, Lackey J, Moussa A, Shah SV, Castellanos AM, Khan S, Schonk M, Thome T, Salyers ZR, Jakkidi N, Kim K, Yang Q, Hepple RT, Ryan TE (2024) Chronic aryl hydrocarbon receptor activity impairs muscle mitochondrial function with tobacco smoking. https://doi.org/10.1002/jcsm.13439 | Mouse | Skeletal muscle |
Al-Sabri 2024 Sci Rep | 2024 | Al-Sabri MH, Ammar N, Korzh S, Alsehli AM, Hosseini K, Fredriksson R, Mwinyi J, Williams MJ, Boukhatmi H, Schiöth HB (2024) Fluvastatin-induced myofibrillar damage is associated with elevated ROS, and impaired fatty acid oxidation, and is preceded by mitochondrial morphological changes. https://doi.org/10.1038/s41598-024-53446-w | Drosophila | Skeletal muscle |
Donnelly 2024 Redox Biol | 2024 | Donnelly C, Komlódi T, Cecatto C, Cardoso LHD, Compagnion A-C, Matera A, Tavernari D, Campiche O, Paolicelli RC, Zanou N, Kayser B, Gnaiger E, Place N (2024) Functional hypoxia reduces mitochondrial calcium uptake. Redox Biol 71:103037. https://doi.org/10.1016/j.redox.2024.103037 | Human Mouse | Heart Skeletal muscle |
Cefis 2024 Acta Physiol (Oxf) | 2024 | Cefis M, Dargegen M, Marcangeli V, Taherkhani S, Dulac M, Leduc-Gaudet JP, Mayaki D, Hussain SNA, Gouspillou G (2024) MFN2 overexpression in skeletal muscles of young and old mice causes a mild hypertrophy without altering mitochondrial respiration and H2O2 emission. Acta Physiol (Oxf) [Epub ahead of print]. https://doi.org/10.1111/apha.14119 | Mouse | Skeletal muscle |
Taylor 2024 Pilot Feasibility Stud | 2024 | Taylor MK, Burns JM, Choi IY, Herda TJ, Lee P, Smith AN, Sullivan DK, Swerdlow RH, Wilkins HM (2024) Protocol for a single-arm, pilot trial of creatine monohydrate supplementation in patients with Alzheimer's disease. Pilot Feasibility Stud 10:42. https://doi.org/10.1186/s40814-024-01469-5 | Human | Lymphocyte Platelet |
Meldau 2024 Mol Genet Metab Rep | 2024 | Meldau S, Ackermann S, Riordan G, van der Watt GF, Spencer C, Raga S, Khan K, Blackhurst DM, van der Westhuizen FH (2024) A novel mitochondrial DNA variant in MT-ND6: m.14430A>C p.(Trp82Gly) identified in a patient with Leigh syndrome and complex I deficiency. Mol Genet Metab Rep 39:101078. https://doi.org/10.1016/j.ymgmr.2024.101078 | Human | Fibroblast |
Sorby-Adams 2024 Redox Biol | 2024 | Sorby-Adams A, Prime TA, Miljkovic JL, Prag HA, Krieg T, Murphy MP (2024) A model of mitochondrial superoxide production during ischaemia-reperfusion injury for therapeutic development and mechanistic understanding. Redox Biol 72:103161. https://doi.org/10.1016/j.redox.2024.103161 | Rat | Heart |
Dominguez-Lopez 2023 Neuropharmacology | 2023 | Dominguez-Lopez S, Ahn B, Sataranatarajan K, Ranjit R, Premkumar P, Van Remmen H, Beckstead MJ (2023) Long-term methamphetamine self-administration increases mesolimbic mitochondrial oxygen consumption and decreases striatal glutathione. https://doi.org/10.1016/j.neuropharm.2023.109436 | Mouse | Nervous system |
Mahmoud 2023 Pharmacol Res | 2023 | Mahmoud AM, Kostrzewa M, Marolda V, Cerasuolo M, Maccarinelli F, Coltrini D, Rezzola S, Giacomini A, Mollica MP, Motta A, Paris D, Zorzano A, Marzo VD, Ronca R, Ligresti A (2023) Cannabidiol alters mitochondrial bioenergetics via VDAC1 and triggers cell death in hormone-refractory prostate cancer. https://doi.org/10.1016/j.phrs.2023.106683 | Mouse | Endothelial;epithelial;mesothelial cell |
Gautam 2023 Neurobiol Dis | 2023 | Gautam M, Genç B, Helmold B, Ahrens A, Kuka J, Makrecka-Kuka M, Günay A, Koçak N, Aguilar-Wickings IR, Keefe D, Zheng G, Swaminathan S, Redmon M, Zariwala HA, Özdinler PH (2023) SBT-272 improves TDP-43 pathology in ALS upper motor neurons by modulating mitochondrial integrity, motility, and function. https://doi.org/10.1016/j.nbd.2023.106022 | Rat | Heart Nervous system |
Donnelly 2023 MitoFit | 2023 | Donnelly C, Komlódi T, Cecatto C, Cardoso LHD, Compagnion AC, Matera A, Tavernari D, Zanou N, Kayser B, Gnaiger E, Place N (2023) Functional hypoxia reduces mitochondrial calcium uptake. MitoFit Preprints 2023.2. https://doi.org/10.26124/mitofit:2023-0002 — 2024-11-17 published in Redox Biol. | Human Mouse | Skeletal muscle Heart Nervous system Other cell lines |
Leduc-Gaudet 2023 Nat Commun | 2023 | Leduc-Gaudet JP, Franco-Romero A, Cefis M, Moamer A, Broering FE, Milan G, Sartori R, Chaffer TJ, Dulac M, Marcangeli V, Mayaki D, Huck L, Shams A, Morais JA, Duchesne E, Lochmuller H, Sandri M, Hussain SNA, Gouspillou G (2023) MYTHO is a novel regulator of skeletal muscle autophagy and integrity. https://doi.org/10.1038/s41467-023-36817-1 | Mouse | Skeletal muscle |
Koizumi 2023 Front Cardiovasc Med | 2023 | Koizumi T, Watanabe M, Yokota T, Tsuda M, Handa H, Koya J, Nishino K, Tatsuta D, Natsui H, Kadosaka T, Koya T, Nakao M, Hagiwara H, Kamada R, Temma T, Tanaka S, Anzai T (2023) Empagliflozin suppresses mitochondrial reactive oxygen species generation and mitigates the inducibility of atrial fibrillation in diabetic rats. Front Cardiovasc Med 10: 1005408. | Rat | Heart |
Salmon 2023 Geroscience | 2023 | Salmón P, Millet C, Selman C, Monaghan P, Dawson NJ (2023) Tissue-specific reductions in mitochondrial efficiency and increased ROS release rates during ageing in zebra finches, Taeniopygia guttata. https://doi.org/10.1007/s11357-022-00624-1 | Birds | Skeletal muscle Liver |
Devaux 2023 J Comp Physiol B | 2023 | Devaux JBL, Hedges CP, Birch N, Herbert N, Renshaw GMC, Hickey AJR (2023) Electron transfer and ROS production in brain mitochondria of intertidal and subtidal triplefin fish (Tripterygiidae). https://doi.org/10.1007/s00360-023-01495-4 | Fishes | Nervous system |
Czyzowska 2023 Redox Biol | 2023 | Czyżowska A, Brown J, Xu H, Sataranatarajan K, Kinter M, Tyrell VJ, O'Donnell VB, Van Remmen H (2023) Elevated phospholipid hydroperoxide glutathione peroxidase (GPX4) expression modulates oxylipin formation and inhibits age-related skeletal muscle atrophy and weakness. https://doi.org/10.1016/j.redox.2023.102761 | Mouse | Skeletal muscle |
Som 2023 Am J Physiol Cell Physiol | 2023 | Som R, Fink BD, Yu L, Sivitz WI (2023) Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression. Am J Physiol Cell Physiol 324:C1236-48. doi: 10.1152/ajpcell.00565.2022 | Mouse | Fat |
Steffen 2023 J Exp Biol | 2023 | Steffen JBM, Sokolov EP, Bock C, Sokolova IM (2023) Combined effects of salinity and intermittent hypoxia on mitochondrial capacity and reactive oxygen species efflux in the Pacific oyster, Crassostrea gigas. https://doi.org/10.1242/jeb.246164 | Molluscs | Lung;gill |
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MiPNet24.10 H2O2 flux analysis | 2021-10-22 | Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4 | ||
MiPNet26.10 MgG data analysis | 2021-09-16 | Measurement of mitochondrial ATP production using Magnesium Green: DL-Protocols and data analysis with DatLab 7.4 | ||
MiPNet17.05 O2k-Fluo LED2-Module | 2021-06-22 | O2k-Fluo LED2-Module. | ||
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MiPNet24.08 Safranin Analysis Template | 2020-05-13 | Excel template for safranin data analysis. | ||
MiPNet25.14 TPP Analysis Template | 2020-##-## | Excel template for TPP data analysis. | ||
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Marquez 2020 J Am Heart Assoc | 2020 | Marquez AM, Morgan RW, Ko T, Landis WP, Hefti MM, Mavroudis CD, McManus MJ, Karlsson M, Starr J, Roberts AL, Lin Y, Nadkarni V, Licht DJ, Berg RA, Sutton RM, Kilbaugh TJ (2020) Oxygen exposure during cardiopulmonary resuscitation is associated with cerebral oxidative injury in a randomized, blinded, controlled, preclinical trial. J Am Heart Assoc 9:015032. | Pig | Nervous system |
Mikulas 2020 Materials | 2020 | Mikulás K, Komlódi T, Földes A, Sváb G, Horváth G, Nagy AM, Ambrus A, Gyulai-Gaál Sz, Gera I, Hermann P, Varga G, Tretter L (2020) Bioenergetic Impairment of triethylene glycol dimethacrylate- (TEGDMA-) treated dental pulp stem cells (DPSCs) and isolated brain mitochondria are amended by redox compound methylene blue. Materials (Basel) 13(16):3472. | Guinea pig Human | Stem cells |
Bhaskaran 2020 Aging Cell | 2020 | Bhaskaran S, Pollock N, C Macpherson P, Ahn B, Piekarz KM, Staunton CA, Brown JL, Qaisar R, Vasilaki A, Richardson A, McArdle A, Jackson MJ, Brooks SV, Van Remmen H (2020) Neuron-specific deletion of CuZnSOD leads to an advanced sarcopenic phenotype in older mice. Aging Cell 19:e13225. | Mouse | Skeletal muscle |
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Falcao-Tebas 2020 J Physiol | 2020 | Falcão-Tebas F, Marin EC, Kuang J, Bishop DJ, McConell GK (2020) Maternal exercise attenuates the lower skeletal muscle glucose uptake and insulin secretion caused by paternal obesity in female adult rat offspring. J Physiol 598:4251-70. | Rat | Skeletal muscle |
Pham 2020 Eur J Appl Physiol | 2020 | Pham T, MacRae CL, Broome SC, D'souza RF, Narang R, Wang HW, Mori TA, Hickey AJR, Mitchell CJ, Merry TL (2020) MitoQ and CoQ10 supplementation mildly suppresses skeletal muscle mitochondrial hydrogen peroxide levels without impacting mitochondrial function in middle-aged men. Eur J Appl Physiol 120:1657-69. | Human | Skeletal muscle |
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Dulac 2020 J Physiol | 2020 | Dulac M, Leduc-Gaudet JP, Reynaud O, Ayoub MB, Guérin A, Finkelchtein M, Hussain SN, Gouspillou G (2020) Drp1 knockdown induces severe muscle atrophy and remodelling, mitochondrial dysfunction, autophagy impairment and denervation . J Physiol 598:3691-710. | Mouse | Skeletal muscle |
Mahul-Mellier 2020 Proc Natl Acad Sci U S A | 2020 | Mahul-Mellier AL, Burtscher J, Maharjan N, Weerens L, Croisier M, Kuttler F, Leleu M, Knott GW, Lashuel HA (2020) The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration. Proc Natl Acad Sci U S A 117:4971-82. | Mouse | Nervous system |
Davidson 2020 Circ Res | 2020 | Davidson MT, Grimsrud P, Lai L, Draper J, Fisher-Wellman KH, Narowski TM, Koves TR, Kelly DP, Muoio DM (2020) Extreme acetylation of the cardiac mitochondrial proteome does not promote heart failure. Circ Res 127:1094-108. | Mouse | Heart |
Hernansanz-Agustin 2020 Nature | 2020 | Hernansanz-Agustín P, Choya-Foces C, Carregal-Romero S, Ramos E, Oliva T, Villa-Piña T, Moreno L, Izquierdo-Álvarez A, Cabrera-García JD, Cortés A, Lechuga-Vieco AV, Jadiya P, Navarro E, Parada E, Palomino-Antolín A, Tello D, Acín-Pérez R, Rodríguez-Aguilera JC, Navas P, Cogolludo Á, López-Montero I, Martínez-Del-Pozo Á, Egea J, López MG, Elrod JW, Ruíz-Cabello J, Bogdanova A, Enríquez JA, Martínez-Ruiz A (2020) Na+ controls hypoxic signalling by the mitochondrial respiratory chain. Nature 586:287-91. | Rat | Heart |
Marrocco 2020 Thesis | 2020 | Marrocco A (2020) Alterations of CCSP expression and macrophages metabolism in the development of silica-induced pulmonary inflammation and fibrosis. PhD Thesis 108. | Mouse | Macrophage-derived |
Souza da Silva 2020 Mol Neurobiol | 2020 | Souza da Silva J, Nonose Y, Rohden F, Lukasewicz Ferreira PC, Fontella FU, Rocha A, Wigner Brochier A, Vieira Apel R, de Lima TM, Seminotti B, Amaral AU, Galina A, Souza DO (2020) Guanosine neuroprotection of presynaptic mitochondrial calcium homeostasis in a mouse study with amyloid-β oligomers. Mol Neurobiol 57:4790-809. | Mouse | Nervous system |
Pharaoh 2020 Sci Rep | 2020 | Pharaoh G, Brown JL, Sataranatarajan K, Kneis P, Bian J, Ranjit R, Hadad N, Georgescu C, Rabinovitch P, Ran Q, Wren JD, Freeman W, Kinter M, Richardson A, Van Remmen H (2020) Targeting cPLA2 derived lipid hydroperoxides as a potential intervention for sarcopenia. Sci Rep 10:13968. | Mouse | Skeletal muscle |
Abid 2020 FASEB J | 2020 | Abid H, Ryan ZC, Delmotte P, Sieck GC, Lanza IR (2020) Extramyocellular interleukin-6 influences skeletal muscle mitochondrial physiology through canonical JAK/STAT signaling pathways. FASEB J 34:14458-72. | Mouse | Skeletal muscle |
MiPNet24.11 mtMP calculation | 2019-08-05 | Mitochondrial membrane potential calculation | ||
MiPNet20.14 AmplexRed H2O2-production | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed. | Mouse | Heart |
MiPNet20.13 Safranin mt-membranepotential | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with safranin or TMRM. | Mouse | Nervous system |
MiPNet18.05 Amplex-Mouse-heart | 2019-06-24 | O2k-Fluorometry: HRR and H2O2 production in mouse cardiac tissue homogenate. | Mouse | Heart |
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Holsgrove 2019 Thesis | 2019 | Holsgrove A (2019) The effect of temperature on cardiac energetics in the rainbow trout, Onchorhynchus mykiss. Phd Thesis 166. | Fishes | Heart |
Ponce 2019 Thesis | 2019 | Ponce JM (2019) Investigating the roles of cyclin C in the mammalian heart. PhD Thesis 137. | Mouse | Heart |
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Devaux 2019 Front Physiol | 2019 | Devaux JBL, Hedges CP, Birch N, Herbert N, Renshaw GMC, Hickey AJR (2019) Acidosis maintains the function of brain mitochondria in hypoxia-tolerant triplefin fish: a strategy to survive acute hypoxic exposure? Front Physiol 9:1941. | Fishes | Nervous system |
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Power 2019 PLoS One | 2019 | Power AS, Norman R, Jones TLM, Hickey AJ, Ward ML (2019) Mitochondrial function remains impaired in the hypertrophied right ventricle of pulmonary hypertensive rats following short duration metoprolol treatment. PLoS One 14:e0214740. | Rat | Heart |
Bundgaard 2019 Sci Rep | 2019 | Bundgaard A, James AM, Gruszczyk AV, Martin J, Murphy MP, Fago A (2019) Metabolic adaptations during extreme anoxia in the turtle heart and their implications for ischemia-reperfusion injury. Sci Rep 9:2850. | Mouse Reptiles | Heart |
Munro 2019 Aging Cell | 2019 | Munro D, Baldy C, Pamenter ME, Treberg JR (2019) The exceptional longevity of the naked mole-rat may be explained by mitochondrial antioxidant defenses. Aging Cell 18:e12916. | Mouse Other mammals | Heart Skeletal muscle |
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Ahn 2019 J Cachexia Sarcopenia Muscle | 2019 | Ahn B, Ranjit R, Premkumar P, Pharaoh G, Piekarz KM, Matsuzaki S, Claflin DR, Riddle K, Judge J, Bhaskaran S, Satara Natarajan K, Barboza E, Wronowski B, Kinter M, Humphries KM, Griffin TM, Freeman WM, Richardson A, Brooks SV, Van Remmen H (2019) Mitochondrial oxidative stress impairs contractile function but paradoxically increases muscle mass via fiber branching. J Cachexia Sarcopenia Muscle 10:411-28. | Mouse | Skeletal muscle |
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Laouafa 2019 Acta Physiol (Oxf) | 2019 | Laouafa S, Roussel D, Marcouiller F, Soliz J, Gozal D, Bairam A, Joseph V (2019) Roles of oestradiol receptor alpha and beta against hypertension and brain mitochondrial dysfunction under intermittent hypoxia in female rats. Acta Physiol (Oxf) 226:e13255. | Rat | Nervous system |
Rajendran 2019 EMBO Mol Med | 2019 | Rajendran J, Purhonen J, Tegelberg S, Smolander OP, Mörgelin M, Rozman J, Gailus-Durner V, Fuchs H, Hrabe de Angelis M, Auvinen P, Mervaala E, Jacobs HT, Szibor M, Fellman V, Kallijärvi J (2019) Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy. EMBO Mol Med 11:e9456. | Mouse | Liver Kidney Heart |
Lefranc 2019 Hypertension | 2019 | Lefranc C, Friederich-Persson M, Braud L, Palacios-Ramirez R, Karlsson S, Boujardine N, Motterlini R, Jaisser F, Nguyen Dinh Cat A (2019) MR (mineralocorticoid receptor) induces adipose tissue senescence and mitochondrial dysfunction leading to vascular dysfunction in obesity. Hypertension 73:458-68. | Mouse | Fat |
Skrivergaard 2019 Viruses | 2019 | Skrivergaard S, Jensen MS, Rolander TB, Nguyen TBN, Bundgaard A, Nejsum LN, Martensen PM (2019) The cellular localization of the p42 and p46 oligoadenylate synthetase 1 isoforms and their impact on mitochondrial respiration. Viruses 11:E1122. | Human | Other cell lines HeLa |
Cooper 2019 Exp Physiol | 2019 | Cooper MA, McCoin C, Pei D, Thyfault JP, Koestler D, Wright DE (2019) Reduced mitochondrial reactive oxygen species production in peripheral nerves of mice fed a ketogenic diet. Exp Physiol 103:1206-12. | Mouse | Nervous system |
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Pharaoh 2019 Mol Neurobiol | 2019 | Pharaoh G, Owen D, Yeganeh A, Premkumar P, Farley J, Bhaskaran S, Ashpole N, Kinter M, Van Remmen H, Logan S (2019) Disparate central and peripheral effects of circulating IGF-1 deficiency on tissue mitochondrial function. Mol Neurobiol 57:1317-31. | Mouse | Skeletal muscle Nervous system Fat |
Cannon 2019 J Appl Physiol (1985) | 2019 | Cannon DT, Rodewohl L, Adams V, Breen EC, Bowen TS (2019) Skeletal myofiber VEGF deficiency leads to mitochondrial, structural and contractile alterations in mouse diaphragm. J Appl Physiol (1985) 127:1360-69. | Mouse | Skeletal muscle |
McMurray 2019 FASEB J | 2019 | McMurray F, MacFarlane M, Kim K, Patten DA, Wei-LaPierre L, Fullerton MD, Harper ME (2019) Maternal diet-induced obesity alters muscle mitochondrial function in offspring without changing insulin sensitivity. FASEB J 33:13515-26. | Mouse | Skeletal muscle |
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Perry 2019 J Mol Cell Cardiol | 2019 | Perry JB, Davis GN, Allen ME, Makrecka-Kuka M, Dambrova M, Grange RW, Shaikh SR, Brown DA (2019) Cardioprotective effects of idebenone do not involve ROS scavenging: Evidence for mitochondrial complex I bypass in ischemia/reperfusion injury. J Mol Cell Cardiol 135:160-171. | Rat | Heart |
Shirakawa 2019 Sci Rep | 2019 | Shirakawa R, Yokota T, Nakajima T, Takada S, Yamane M, Furihata T, Maekawa S, Nambu H, Katayama T, Fukushima A, Saito A, Ishimori N, Dela F, Kinugawa S, Anzai T (2019) Mitochondrial reactive oxygen species generation in blood cells is associated with disease severity and exercise intolerance in heart failure patients. Sci Rep 9:14709. | Human | Blood cells |
Ruegsegger 2019 JCI Insight | 2019 | Ruegsegger GN, Vanderboom PM, Dasari S, Klaus KA, Kabiraj P, McCarthy CB, Lucchinetti CF, Nair KS (2019) Exercise and metformin counteract altered mitochondrial function in the insulin-resistant brain. JCI Insight 4:130681. | Mouse | Nervous system |
Axton 2019 J Nutr | 2019 | Axton ER, Beaver LM, St Mary L, Truong L, Logan CR, Spagnoli S, Prater MC, Keller RM, Garcia-Jaramillo M, Ehrlicher SE, Stierwalt HD, Newsom SA, Robinson MM, Tanguay RL, Stevens JF, Hord NG (2019) Treatment with nitrate, but not nitrite, lowers the oxygen cost of exercise and decreases glycolytic intermediates while increasing fatty acid metabolites in exercised zebrafish. J Nutr 00:1–13. | Zebrafish | Skeletal muscle |
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Logan 2018 Thesis | 2018 | Logan C (2018) Nitrate and nitrite differentially affect respiration in zebrafish during exercise. Honors Baccalaureate of Science in Nutrition p44. | Zebrafish | Skeletal muscle |
Kamunde 2018 Free Radic Biol Med | 2018 | Kamunde C, Sharaf M, MacDonald N (2018) H2O2 metabolism in liver and heart mitochondria: Low emitting-high scavenging and high emitting-low scavenging systems. Free Radic Biol Med 124:135-48. | Fishes | Heart Liver |
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Kim 2018 Free Radic Biol Med | 2018 | Kim M, Stepanova A, Niatsetskaya Z, Sosunov S, Arndt S, Murphy MP, Galkin A, Ten VS (2018) Attenuation of oxidative damage by targeting mitochondrial complex I in neonatal hypoxic-ischemic brain injury. Free Radic Biol Med 124:517-24. | Mouse | Nervous system |
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Kukat 2014 PLoS Genet | 2014 | Kukat A, Dogan SA, Edgar D, Mourier A, Jacoby C, Maiti P, Mauer J, Becker C, Senft K, Wibom R, Kudin AP, Hultenby K, Flögel U, Rosenkranz S, Ricquier D, Kunz WS, Trifunovic A (2014) Loss of UCP2 attenuates mitochondrial dysfunction without altering ROS production and uncoupling activity. https://doi.org/10.1371/journal.pgen.1004385 | Mouse | Heart |
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Reilly 2013 J Exp Biol | 2013 | Reilly BD, Hickey AJ, Cramp RL, Franklin CE (2013) Decreased hydrogen peroxide production and mitochondrial respiration in skeletal muscle but not cardiac muscle of the green-striped burrowing frog, a natural model of muscle disuse. J Exp Biol 217:1087-93. | Amphibians | Heart Skeletal muscle |
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Iftikar 2013 PLoS One | 2013 | Iftikar FI, Hickey AJ (2013) Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in Notolabrus celidotus. PLoS One 8:e64120. | Fishes | Heart |
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Gnaiger 2012 Mitochondr Physiol Network Bioblast 2012 | 2012 | Gnaiger E, Meissner B, Laner V, eds (2012) Bioblast 2012: Mitochondrial Competence. Mitochondr Physiol Network 17.12. Oroboros MiPNet Publications, Innsbruck:96 pp. ISBN 978-3-9502399-5-9 | ||
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