Cell count and normalization in HRR: Difference between revisions

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high-resolution terminology - matching measurements at high-resolution

Cell count and normalization in HRR

Description

Cell count N_ce is the number of cells, expressed in the unit [x] (1 Mx = 10⁶ x). Normalization of respiratory rate by cell count yields oxygen flow I_O₂ expressed in units [amol·s^-1·x^-1] (=10^-18 mol·s^-1·x^-1).

Abbreviation: N_ce

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 σ	V_σ			mL	The symbol σ 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_σ.
Cell count in V_σ	N_ce,σ	=	C_ce,σ∙V_σ	mL	The total cell harvest available for respirometry and additional analyses of this sample of cells.
Cell-count concentration in cell suspension V_σ	C_ce,σ	=	N_ce,σ/V_σ	mL
Subsample from V_σ for cell counting	V_σ→N			mL	For reliable normalization of respiration, V_σ→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.
Subsample from V_σ for respirometry J	V_σ→J	=	V_σ-V_σ→N	mL	.

Experimental chamber volume	V			mL	Effective aqueous volume in the experimental system (2.0 mL or 0.5 mL)
Experimental chamber volume + V_{Stopper capillary}	V'	=	V+V_{Stopper capillary}	mL	2.085 or 0.54 mL
Experimental chamber volume + V_{Stopper capillary} + subsample volume	V''	=	V'+V_subsamples	mL	Subsamples taken from the O2k chamber prior to closing the chamber.
Cell-count in experimental chamber	N_ce	=	N·U_ce	x	10⁶ x = 1 Mx
Cell-count concentration in experimental chamber	C_ce	=	N_ce/V	Mx/mL	10⁶ x/mL = 1 Mx/mL = 10⁹ x/L = 1 Gx/L

Volume of respirometric stock J	V_J	=	V_s→J + ΔV_J	mL

MitoPedia methods: Respirometry

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

@@ Line 7: / Line 7: @@
   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.
+:::: {| class="wikitable"
+|-
+! Term !! Symbol !! = !! Definition !! Unit !! Comment
+|-
+| Volume of cell suspension σ || ''V''<sub>σ</sub> ||  ||  || mL || The symbol σ 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>σ</sub>.
+|-
+| Cell count in ''V''<sub>σ</sub> || ''N''<sub>ce,σ</sub> || = || ''C''<sub>ce,σ</sub>∙''V''<sub>σ</sub> || mL || The total cell harvest available for respirometry and additional analyses of this sample of cells.
+|-
+| Cell-count concentration in cell suspension ''V''<sub>σ</sub> || ''C''<sub>ce,σ</sub> || = || ''N''<sub>ce,σ</sub>/''V''<sub>σ</sub> || mL ||
+|-
+| Subsample from ''V''<sub>σ</sub> for cell counting || ''V''<sub>σ→''N''</sub> ||  ||  || mL || For reliable normalization of respiration, ''V''<sub>σ→N</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.
+|-
+| Subsample from ''V''<sub>σ</sub> for respirometry ''J'' || ''V''<sub>σ→''J''</sub> || = || ''V''<sub>σ</sub>-''V''<sub>σ→''N''</sub> || mL || .
+|-
+|  ||  ||  ||  ||  ||
+|-
+| Experimental chamber volume || ''V'' ||  ||  || mL || Effective aqueous volume in the experimental system (2.0 mL or 0.5 mL)
+|-
+| Experimental chamber volume + ''V''<sub>Stopper capillary</sub> || ''V''' || = || ''V''+''V''<sub>Stopper capillary</sub> || mL || 2.085 or 0.54 mL
+|-
+| 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.
+|-
+| Cell-count in experimental chamber || ''N''<sub>ce</sub> || = || ''N''·''U''<sub>ce</sub> || x || 10<sup>6</sup> x = 1 Mx
+|-
+| 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
+|-
+|  ||  ||  ||  ||  ||
+|-
+| Volume of respirometric stock ''J'' || ''V''<sub>''J''</sub> || = || ''V''<sub>s→J</sub> + Δ''V''<sub>''J''</sub> || mL ||
+|-
+|}