Cell count and normalization in HRR: Difference between revisions
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ย Communicated by [[Gnaiger Erich]] 2020-09-18 | ย Communicated by [[Gnaiger Erich]] 2020-09-18 | ||
== Cell count and cell concentration == | == Cell count and cell concentration == | ||
== Cell concentration from cell suspension to experimental chamber == | == Cell concentration from cell suspension to experimental chamber == | ||
:::: Table 1. Definition of terms, symbols and units. | |||
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! Term !! Symbol !! = !! Definition !! Unit !! Comment | |||
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| Volume of cell suspension ฯ || ''V''<sub>ฯ</sub> ||ย ||ย || mL || The symbol ฯ is used for 'suspension', distinguished from the symbol s for 'sample'. Frequently the sediment of cells obtained from centrifugation is re-suspended in the experimental medium (''e.g.'' MiR05 plus pyruvate) to obtain ''V''<sub>ฯ</sub>. | |||
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| Cell count in ''V''<sub>ฯ</sub> || ''N''<sub>ce,ฯ</sub> || = || ''C''<sub>ce,ฯ</sub>โ''V''<sub>ฯ</sub> || mL || The total cell harvest available for respirometry and additional analyses of this sample of cells. | |||
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| Cell-count concentration in cell suspension ''V''<sub>ฯ</sub> || ''C''<sub>ce,ฯ</sub> || = || ''N''<sub>ce,ฯ</sub>/''V''<sub>ฯ</sub> || mL || | |||
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| Subsample from ''V''<sub>ฯ</sub> for cell counting || ''V''<sub>ฯโ''N''</sub> ||ย ||ย || mL || For reliable normalization of respiration, ''V''<sub>ฯโN</sub> 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. | |||
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| Subsample from ''V''<sub>ฯ</sub> for respirometry ''J'' || ''V''<sub>ฯโ''J''</sub> || = || ''V''<sub>ฯ</sub>-''V''<sub>ฯโ''N''</sub> || mL || . | |||
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|ย ||ย ||ย ||ย ||ย || | |||
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| Experimental chamber volume || ''V'' ||ย ||ย || mL || Effective aqueous volume in the experimental system (2.0 mL or 0.5 mL) | |||
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| Experimental chamber volume + ''V''<sub>Stopper capillary</sub> || ''V''' || = || ''V''+''V''<sub>Stopper capillary</sub> || mL || 2.085 or 0.54 mL | |||
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| Experimental chamber volume + ''V''<sub>Stopper capillary</sub> + subsample volume || ''V'''' || = || ''V'''+''V''<sub>subsamples</sub> || mL || Subsamples taken from the O2k chamber prior to closing the chamber. | |||
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| Cell-count in experimental chamber || ''N''<sub>ce</sub> || = || ''N''ยท''U''<sub>ce</sub> || x || 10<sup>6</sup> x = 1 Mx | |||
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| Cell-count concentration in experimental chamber || ''C''<sub>ce</sub> || = || ''N''<sub>ce</sub>/''V'' || Mx/mL || 10<sup>6</sup> x/mL = 1 Mx/mL = 10<sup>9</sup> x/L = 1 Gx/L | |||
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|ย ||ย ||ย ||ย ||ย || | |||
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| Volume of respirometric stock ''J'' || ''V''<sub>''J''</sub> || = || ''V''<sub>sโJ</sub> + ฮ''V''<sub>''J''</sub> || mL || | |||
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Revision as of 21:53, 17 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 [amolยทs-1ยทx-1] (=10-18 molยทs-1ยทx-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
Cell concentration from cell suspension to experimental chamber
- Table 1. Definition of terms, symbols and units.
Term Symbol = Definition Unit Comment Volume of cell suspension ฯ Vฯ mL The symbol ฯ is used for 'suspension', distinguished from the symbol s for 'sample'. Frequently the sediment of cells obtained from centrifugation is re-suspended in the experimental medium (e.g. MiR05 plus pyruvate) to obtain Vฯ. Cell count in Vฯ Nce,ฯ = Cce,ฯโVฯ mL The total cell harvest available for respirometry and additional analyses of this sample of cells. Cell-count concentration in cell suspension Vฯ Cce,ฯ = Nce,ฯ/Vฯ mL Subsample from Vฯ for cell counting VฯโN mL For reliable normalization of respiration, VฯโN 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. Subsample from Vฯ for respirometry J VฯโJ = Vฯ-VฯโN mL . Experimental chamber volume V mL Effective aqueous volume in the experimental system (2.0 mL or 0.5 mL) Experimental chamber volume + VStopper capillary V' = V+VStopper capillary mL 2.085 or 0.54 mL Experimental chamber volume + VStopper capillary + subsample volume V'' = V'+Vsubsamples mL Subsamples taken from the O2k chamber prior to closing the chamber. Cell-count in experimental chamber Nce = NยทUce x 106 x = 1 Mx Cell-count concentration in experimental chamber Cce = Nce/V Mx/mL 106 x/mL = 1 Mx/mL = 109 x/L = 1 Gx/L Volume of respirometric stock J VJ = VsโJ + ฮVJ mL
MitoPedia methods: Respirometry
MitoPedia O2k and high-resolution respirometry:
DatLab,
O2k-Respirometry,
O2k-FluoRespirometry