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Difference between revisions of "Cell count and normalization in HRR"

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|abbr=''N''<sub>ce</sub>
|abbr=''N''<sub>ce</sub>
|description=Cell count ''N''<sub>ce</sub> is the number of cells, expressed in the unit [x] (1 Mx = 10<sup>6</sup> x). Normalization of respiratory rate by ''cell count'' yields oxygen ''flow'' ''I''<sub>O<sub>2</sub></sub> expressed in units [amol·s<sup>-1</sup>·x<sup>-1</sup>] (=10<sup>-18</sup> mol·s<sup>-1</sup>·x<sup>-1</sup>).
|description=Cell count ''N''<sub>ce</sub> is the number of cells, expressed in the unit [x] (1 Mx = 10<sup>6</sup> x). Normalization of respiratory rate by ''cell count'' yields oxygen ''flow'' ''I''<sub>O<sub>2</sub></sub> expressed in units [amol·s<sup>-1</sup>·x<sup>-1</sup>] (=10<sup>-18</sup> mol·s<sup>-1</sup>·x<sup>-1</sup>).
|info=[[BEC 2020.1 doi10.26124bec2020-0001.v1]], [[Gnaiger 2020 MitoPathways]]
|info=[[Gnaiger 2020 MitoPathways]]
}}
}}
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Revision as of 15:31, 18 September 2020


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: 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

Tube Rack with Stopper.jpg
Subsamples are transferred from the stock cell suspensions into the O2k chamber in four possible ways:
1. Complete replacement
The stock cell-count concentration Cce,J is equal to the initial cell-count concentration Cce in the experimental chamber. The stopper has to be removed from the O2k chamber and placed into an empty 50-mL Falcon on the Tube Rack (Figure on the right). Aqueous medium is siphoned off from the chamber and discharged. A volume VJ.i equal or higher than V' (experimental chamber volume plus volume of stopper capillary) is added into the empty chamber. The stirrer may be left on, but is switched off if this helps to avoid foam formation. The stirrer must be switched on before closing the chamber with the stopper.
2. Pipetting into V'-VJ.i
After the O2k chamber filled with experimental respiration medium is closed with the stopper in the volume-calibrated position, any excess medium is siphoned off the receptacle of the stopper. Then the stopper is gently removed from the chamber and placed into an empty 50-mL Falcon on the Tube Rack (Figure on the right). A volume VJ.i of respiration medium is pipetted off the chamber and discharged. The same volume VJ.i of concentrated stock cell suspension VJ is pipetted into the chamber. Then the total volume is V'. When the chamber is closed with the stopper, the experimental volume is V, and the stopper capillary is full with experimental medium, if no drops are lost from the removed stopper and no medium is lost by evaporation.
3. Pipetting into V'
After the O2k chamber filled with experimental respiration medium is closed with the stopper in the volume-calibrated position, any excess medium is siphoned off the receptacle of the stopper. Then the stopper is gently removed from the chamber and placed into an empty 50-mL Falcon on the Tube Rack (Figure on the right). A volume VJ.i of concentrated stock cell suspension VJ is pipetted into the chamber. Then the total volume is V'+VJ.i. When the chamber is closed with the stopper, the experimental volume is V, and the stopper capillary is full with experimental medium and an excess of VJ.i is siphoned off the receptacle of the stopper. In this way more cells are lost compared to the previous procedure, but an excess volume is available that may be helpful in case of having to remove any trapped gas bubble from the chamber.
4. Titration into closed chamber
After the O2k chamber filled with experimental respiration medium is closed with the stopper in the volume-calibrated position, a volume VJ.i of concentrated stock cell suspension VJ is titrated into the chamber, identical to adding a suspension of isolated mitochondria. This approach is well tested for yeast cells, but is not recommended for mammalian cells which may loose viability due to the high shear stress in a Hamilton syringe. When the titration is performed fast into the O2k chamber, a volume VJ.i of pure medium is extruded through the stopper, before mixing of cells into the chamber volume, such that no cells are lost from the experimental chamber. The dilution of the cell stock concentration in the experimental chamber is then calculated with reference to the chamber volume V. Slow titrations, however, entail an excape of cells into the stopper capillary leading to an inaccurately calculated cell-count concentration Cce.


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.
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. VJ = Vs→J + ΔVJ
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
Respirometric experiment
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


MitoPedia methods: Respirometry 


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