Intact cells

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MitoPedia

Intact cells

Description

Intact cells or viable cells (vce) are characterized by an intact cell membrane. Cell viability should be >95% for various experimental investigations, including cell respirometry. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce). In contrast, the cell membrane of intact cells can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry.

Abbreviation: vce

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

HRR and intact 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 signalling (particularly high [Ca2+]). Contidions during respiratory measurement can then be maintained close to cell culture conditions.
Respiration of intact 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 intact 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.


Hepatocytes

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



MitoPedia methods: Respirometry