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Cell count and normalization in HRR

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Revision as of 12:44, 18 September 2020 by Gnaiger Erich (talk | contribs)


high-resolution terminology - matching measurements at high-resolution


Cell count and normalization in HRR

Description

Cell count Nce is the number of cells, expressed in the unit [x] (1 Mx = 106 x). Normalization of respiratory rate by cell count yields oxygen flow IO2 expressed in units [amol·s-1·x-1] (=10-18 mol·s-1·x-1).

Abbreviation: Nce

Reference: BEC 2020.1 doi10.26124bec2020-0001.v1, Gnaiger 2020 MitoPathways

Communicated by Gnaiger Erich 2020-09-18

Cell count and cell concentration

V' is 7 % and 4 % larger than V in the 0.5 mL and 2.0 mL chamber, respectively. If cells are pipetted into the O2k chamber opened after closing and siphoning off any medium from the receptacle of the stopper, the final cell-count concentration in the respirometric chamber would be underestimated, if the volume of the stopper capillary is ignored in the calculation of cell-count concentration.

Cell concentration from cell suspension to experimental chamber

Oxygen flow IO2 [amol·s-1·x-1], normalized for the cell count Nce [x], is as inaccurate and irreproducible as the cell count. In technical respirometric repeats with the same volume of cells sampled from the same stock, reproducibility of volume-specific oxygen flux JO2 [pmol·s-1·mL-1] depends on the homogeneity of the cell stock and reproducibility of titration of the cell suspension into the experimental chamber. If cell counts are obtained for subsamples obtained from each experimental chamber separately, the variability of IO2 and JO2 can be compared to assess the reproducibility of the respirometric measurement versus cell counting.
Work in progress
Table 1. Definition of terms, symbols and units.
Quantity Term Symbol = Definition Unit Comment
Cell suspension σ σ
V volume of cell suspension σ Vσ mL The symbol σ is used for 'suspension', distinguished from the symbol s for 'sample'. Cell suspension σ is the first suspension obtained upon harvesting the cells. Frequently the sediment of cells obtained from centrifugation, with Vs, is re-suspended in the experimental medium (e.g. MiR05 plus pyruvate) to obtain Vσ = Vs + ΔVσ.
NX cell count in Vσ Nce,σ = Cce,σVσ Mx The total cell harvest available for respirometry and additional analyses of this sample of cells.
C cell-count concentration in cell suspension Vσ Cce,σ = Nce,σ/Vσ Mx/mL
V subsample from Vσ for cell counting Vσ→N mL For a reliable cell count and normalization of respiration, Vσ→N should be equal or larger than 0.05 mL. Smaller volumes are taken as a subsample for counting, if the aim is merely to define a procedure of dilutions, with final subsamples taken after dilutions for cell-counting.
V subsample from Vσ for respirometry J Vσ→J = Vσ-Vσ→N mL For high accuracy and reproducibility of technical repeats of respiration, Vσ→J should be equal or larger than 0.05 mL.
NX cell count in cell-count subsample from σ Nce,σ→N = Cce,σVσ→N Mx These cells are not available for respirometry, but only a fraction is required for addition into cell counters, hence several technical repeats of cell counting are feasible, or remaing cells may be used for additional characterization of the cell sample.
Respirometric stock J J
V Volume added to Vσ→J ΔVJ = VJ-Vσ→J mL Volume added for dilution of Cce,σ to Cce,J.
V volume of respirometric stock J VJ = Nce,J/Cce,J mL The respirometric stock is the cell suspension diluted from Vσ to VJ for further subsampling of cells to be added into the respirometric chamber for measurement of flux J.
NX cell count in VJ Nce,J = Cce,σVσ→J = Nce,σ-Nce,σ→N Mx The cell count available for respirometry.
C cell-count concentration respirometric stock VJ Cce,J = Mx/mL If cells are pipetted into the O2k chamber opened after closing and siphoning off any medium from the receptacle of the stopper, then Cce,J is adjusted according to Cce·V'/VJ.i.
V subsample volume of stock VJ titrated into respirometer chamber VJ.i mL i = 1 to n, where n is the number of technical respirometric repeats. For a reliable cell count and normalization of respiration, VJ.i should be equal or larger than 0.05 mL.
ratio maximum number of technical JO2 repeats n = VJ/VJ.i
V experimental chamber volume V mL Effective aqueous volume in the experimental system (2.0 mL or 0.5 mL)
V experimental chamber volume + VStopper capillary V' = V+VStopper capillary mL 2.085 or 0.54 mL. The volume of the stopper capillary does not mix with V in the closed chamber. Therefore, only V is the experimental volume and VStopper capillary is considered as a 'dead volume' outside of the experimental chamber volume.
V experimental chamber volume + VStopper capillary + subsample volume V'' = V'+Vsubsamples mL Subsamples taken from the O2k chamber prior to closing the chamber.
NX cell-count in experimental chamber Nce = N·Uce Mx 106 x = 1 Mx
C cell-count concentration in experimental chamber Cce = Nce/V Mx/mL 106 x/mL = 1 Mx/mL = 109 x/L = 1 Gx/L
NX cell-count in V' Nce' = Cce·V' Mx
V volume of respirometric stock J VJ = Vs→J + ΔVJ mL


MitoPedia methods: Respirometry 


MitoPedia O2k and high-resolution respirometry: DatLab, Oroboros QM, O2k-Respirometry, O2k-FluoRespirometry