Living cells

high-resolution terminology - matching measurements at high-resolution

Living cells

Description

Cell viability in living cells should be >95 % for various experimental investigations, including cell respirometry. Viable cells (vce) are characterized by an intact plasma membrane barrier function. The total cell count (N_ce) is the sum of viable cells (N_vce) and dead cells (N_dce). In contrast, the plasma membrane can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry. Living cells are frequently labelled as intact cells in the sense of the total cell count, but intact may suggest dual meanings of viable or unaffected by a disease or mitochondrial injury.

Abbreviation: ce

Reference: BEC 2020.1, Gnaiger 2020 BEC MitoPathways, MiPNet08.09 CellRespiration

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HRR and living cells

For details, see

Gnaiger 2020 BEC MitoPathways
Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. - »Bioblast link«.
Cell ergometry
O2k-Publications: Living cells

Respiration medium

The choice of respiratory medium depends on the scientific question and the applied protocol. The advantage of cell culture media is the availability of substrates (e.g. glucose, glutamine), appropriate ionic composition for maintaining the plasma membrane potential and intact signaling (particularly high [Ca²⁺]). Conditions during respiratory measurement can then be maintained close to cell culture conditions.

Respiration of living cells may be measured in mitochondrial respiration medium (e.g. MiR05) followed by permeabilization of the plasma membrane by digitonin for application of mitochondria-targeted SUIT (substrate-uncoupler-inhibitor titration) protocols. Measuring respiration of permeabilized cells, allowing direct access to the mitochondria, is not possible in cell culture media. These media contain high Ca²⁺ concentrations, important for cell physiology as an extracellular concentration, but damaging for mitochondria which swell and disrupt after permeabilization of the plasma membrane at high Ca²⁺ concentrations.

Respiratory states

ROUTINE and LEAK respiration, Electron transfer pathway capacity and ROX can be determined in viable cells (see Gnaiger 2020 BEC MitoPathways). These respiratory coupling states can be evaluated (1) in the absence of external substrates on the basis of internal substrate stores (endogenous respiration), (2) in the presence of specific fuel substrates, or (3) in complex culture media.

Adherent cells

In most cases, adherent cells grown as a monolayer are detached from the culture plate (scrapping or trypsinizing), centrifuged and resuspended for HRR. For more information, see: Attached cells

Appropriate cell-count concentration for HRR

The appropriate cell-count concentration is cell type and cell size dependent. For more information, see: Sample concentration

For ROUTINE, we typically recommend aiming at an O₂ flux of at least 20 pmol·s^-1·mL^-1, up to 40 pmol·s^-1·mL^-1. However, for cell types with low oxygen fluxes (e.g., PBMCs and platelets), ROUTINE respiration in the range of 10–30 pmol·s^-1·mL^-1 may be used.

ROUTINE respiration per cell may depend on cell concentration:

Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. - »Bioblast link«

The units of the cell count and cell-count concentration

The elementary entity of the cell count is the single individual cell or unit cell U_ce. The abstract unit of the quantity count is the elementary unit x with the meaning of 'one item'. A count is the product of a number and the elementary entity, thus the cell count is N_ce = N·U_ce [x]. A cell count of 1 million cells is designated as N_ce = 1·10⁶ x = 1 Mx (M = mega = 10⁶).
Cell concentration can be expressed in terms of various quantities, i.e. cell-mass concentration C_{m_ce} [g·L^-1 = mg·mL^-1], cell-volume concentration C_{V_ce} [mL·L^-1 = µL·mL^-1], or cell-count concentration C_ce [Gx·L^-1 = Mx·mL^-1] (G=10⁹; M=10⁶). A cell-count concentration C_ce of 1.0 Mx·mL^-1 equals 10⁶ x·mL^-1 = 1.0 million cells per milliliter.

» Gnaiger MitoFit Preprints 2020.4, BEC 2020.1 doi10.26124bec2020-0001.v1

Fibroblasts, HUVEC, thymocytes, lymphocytes

A cell-count concentration C_ce of 1.0 Mx·mL^-1 is recommended for many cultured cells, including different cancer or immortalized cell lines. A minimum of 0.1 Mx·mL^-1 is required in many cell types.

Hepatocytes

Isolated hepatocytes are quite large, therefore, <0.1 Mx·mL^-1 can be applied.

Human peripheral blood mononuclear cells

2.0 Mx·mL^-1 is recommended for isolated PBMC. For more information see: »MiPNet21.17«

Human platelets

100 Mx·mL^-1 is recommended for isolated platelets. For more information see: »MiPNet21.17«

Cell viability assessment

Viability assays are used to measure the proportion of viable cells after a potentially traumatic procedure, such as primary disaggregation, cell separation, or cryopreservation. Most viability tests rely on a breakdown in membrane integrity measured by the uptake of a dye to which the cell is normally impermeable (e.g., Trypan Blue) or the release of a dye normally taken up and retained by viable cells (e.g., acridine orange & propidium iodide).

Trypan blue

Trypan blue is a dye used for vital staining. The reactivity of trypan blue is based on the fact that the chromophore is negatively charged and does not interact with the cell nucleus unless the membrane is damaged. Therefore, all cells which exclude the dye are viable.

Acridine orange & propidium iodide

Acridine orange is an intercalating dye that can permeate both live and dead cells. Acridine orange will stain all nucleated cells to generate green fluorescence. Propidium iodide can only enter dead cells with poor membrane integrity so it will stain all dead nucleated cells to generate red fluorescence. Cells stained with both acridine orange and propidium iodide fluoresce red due to quenching, so all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red.

SUITbrowser question: Cell viability test

Plasma membrane intactness of living cells can be assessed by SUIT protocols with the use of substrates that are not plasma membrane permeant. With further chemical permeabilization of the plasma membranes, it is possible to determine the respirometric viability index, assuming that both viable and dead cells contain functional mitochondria.

The SUITbrowser can be used to find SUIT protocols for testing cell viability and other research questions.

References

Bioblast link	Reference	Year
Gnaiger 2020 BEC MitoPathways	Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5^th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002	2020
BEC 2020.1 doi10.26124bec2020-0001.v1	Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1	2020

Oroboros quality management from the O2k to NextGen-O2k
Gnaiger Erich 2020-09-07

MitoPedia methods: Respirometry

MitoPedia O2k and high-resolution respirometry: O2k-Open Support

MitoPedia topics: Sample preparation