Microwell respirometry: Difference between revisions
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== | == Oroboros comments on microplate respirometry == | ||
ย '''In respirometry, high throughput is ''not equivalent to high output''.''' | ย '''In respirometry, high throughput is ''not equivalent to high output''.''' | ||
::ยป [[O2k specifications]] | ::ยป [[O2k specifications]] | ||
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<big>'''Blood cells stressed in 96-well plate'''</big> | <big>'''Blood cells stressed in 96-well plate'''</big> | ||
* In contrast, Keane et al (2015) <ref> Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723โ33. [[Keane 2015 Clin Sci (Lond) |ยปBioblast linkยซ]] </ref> found mitochondrial injuries after cryopreservation of blood cells. The authors state: ''The Seahorse XFe96 Flux analyzer was used according to manufacturer's instructions. .. In brief, cells were seeded into 96-well plates at a density of 3.5 x 10<sup>5</sup> cells/well and allowed to '''adhere overnight. .. then incubated for 60 min at 37 ยฐC in an non-CO<sub>2</sub> incubator, to allow the cells to become equilibrated with the new medium.''''' The reductions in [[ROUTINE respiration]], [[ | * In contrast, Keane et al (2015) <ref> Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723โ33. [[Keane 2015 Clin Sci (Lond) |ยปBioblast linkยซ]] </ref> found mitochondrial injuries after cryopreservation of blood cells. The authors state: ''The Seahorse XFe96 Flux analyzer was used according to manufacturer's instructions. .. In brief, cells were seeded into 96-well plates at a density of 3.5 x 10<sup>5</sup> cells/well and allowed to '''adhere overnight. .. then incubated for 60 min at 37 ยฐC in an non-CO<sub>2</sub> incubator, to allow the cells to become equilibrated with the new medium.''''' The reductions in [[ROUTINE respiration]], [[ET capacity]] and coupling after 1 month cryopreservation may be related to | ||
::(1) measurement on blood cells adherent in microwells; | ::(1) measurement on blood cells adherent in microwells; | ||
::(2) delayed measurement following overnight plus 60 min stress condition. | ::(2) delayed measurement following overnight plus 60 min stress condition. |
Latest revision as of 15:16, 8 June 2020
Microwell respirometry
Oroboros comments on microplate respirometry
In respirometry, high throughput is not equivalent to high output.
Respirometry with cryopreserved blood cells
Successful O2k application
O2k high-resolution respirometry has been successfully applied on cryopreserved cells from human blood samples to detect alterations reflecting human systemic physiological disease. Using blood samples for OXPHOS analysis extends the scope of diagnostic applications of the O2k, when compared to muscle biopsies. Respiration of blood cells is measured with cells in suspension, using the O2k technology. This reduces stress conditions for the cells compared to cells seeded on microplates.
Reports on this successful application of cryopreserved blood cells with O2k high-resolution respirometry can be found by Karabatsiakis and colleagues (2014) [1] which are based on
- (1) functional assays with blood cells in suspension;
- (2) immediate measurement in the OROBOROS O2k after thawing upon cryopreservation.
Highlights of these recent developments:
Blood cells stressed in 96-well plate
- In contrast, Keane et al (2015) [2] found mitochondrial injuries after cryopreservation of blood cells. The authors state: The Seahorse XFe96 Flux analyzer was used according to manufacturer's instructions. .. In brief, cells were seeded into 96-well plates at a density of 3.5 x 105 cells/well and allowed to adhere overnight. .. then incubated for 60 min at 37 ยฐC in an non-CO2 incubator, to allow the cells to become equilibrated with the new medium. The reductions in ROUTINE respiration, ET capacity and coupling after 1 month cryopreservation may be related to
- (1) measurement on blood cells adherent in microwells;
- (2) delayed measurement following overnight plus 60 min stress condition.
References
- โ Karabatsiakis A, Bock C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa I-T (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Translational Psychiatry 4:e397. ยปBioblast linkยซ
- โ Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723โ33. ยปBioblast linkยซ
Are microwell cell cultures physiological?
Biologists go to great lengths to figure out just the right cocktail of chemicals on which to propagate the cells they want to study. Not too much nitrogenโjust the right amount of sulfurโand so on. But then the whole mix is thrown into a flat-bottomed microwell where the newly sprouted cells bunch up and do not exactly look or behave like cells grown au naturel. Hoping to give lab-cultured cells the chance to more closely resemble their in vivo brethren, materials science researchers have designed a technique for growing cells in three dimensions on top of a cushion of air.
- From BioTechniques Newsletter 2015-05-28
- Goral VN et al (2015) Methods for advanced hepatocyte cell culture in microwells utilizing air bubbles. Lab Chip 15:1032-7.