Living cells

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Living cells


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. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce). In contrast, the cell membrane of cells 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 the alternative meaning of viable.

Abbreviation: vce

Reference: Gnaiger 2019 MitoFit Preprint Arch, MiPNet08.09 CellRespiration

HRR and living cells

For details, see

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 cell membrane potential and intact signaling (particularly high [Ca2+]). Conditions during respiratory measurement can then be maintained close to cell culture conditions.
Respiration of viable cells may be measured in mitochondrial respiration medium (e.g. MiR06) followed by permeabilization of the cell membrane by digitonin and applying complex 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, but damaging for mitochondria, which swell and disrupt.

Respiratory states

ROUTINE and LEAK respiration, ET-pathway capacity and ROX can be determined in viable cells (see Gnaiger 2014 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

The lab of Gregory Brewer developed techniques for high-resolution respirometry with the OROBOROS-O2k of neuronal cells attached to a substrate: Attached cells
In most cases, adherent cells grown as a monolayer are detached from the culture plate (scrapping or trypsinizing), centrifuged and resuspended for HRR.

Appropriate cell density for HRR

The appropriate cell number is cell type and cell size dependent. The sample concentration should be high enough to get a reliable respiration even when mitochondria have a low respiratory activity. On the other hand, if respiration is too high, re-oxygenations have to be performed frequently disturbing the experimental course. As a general guideline:
  • Maximum flux up to 100 to 150 pmol.s-1.mL-1
  • Minimum fluxes at 5 pmol.s-1.mL-1
ROUTINE respiration per cell may depend on cell density:
  • 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«

Fibroblasts, HUVEC, thymocytes, lymphocytes

1.0 million cells/mL is recommended for many cultured cells, including different cancer or immortalized cell lines. A minimum of 0.1 million cells/mL is required.


Isolated hepatocytes are quite large, therefore, <0.1 million cells/mL can be applied.

MitoPedia methods: Respirometry 

Cell viability assessment

Work in progress

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 vital dye. The reactivity of trypan blue is based on the fact that the chromophore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the 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

Work in progress

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