Description

Intact cells are characterized by a cell membrane that is impermeable to various dyes, such a trypan blue.

Abbreviation: Not applicable

MitoPedia methods: Respirometry

HRR and intact cells

For primary information, see

Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.
O2k-Publications:_Intact_Cell;_Cultured;_Primary

Respiratory medium

The choice of respiratory medium depends on the scientific question and the applied protocol. The advantage of cell culture medium is the availability of substrates (e.g. glucose, glutamine), appropriate ionic composition for maintaining the cell membrane potential and intact signalling. Contidions during respiratory measurement can then be maintained close to cell culture conditions.

Respiration of intact cells may be measured in 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, ETS capacity and ROX can be determined in intact cells (see MiPNet12.15 RespiratoryStates). These respiratory coupling states can be evaluated in the absence of external substrates on the basis of internal substrate stores (endogenous respiration).

Adherent cells

In general adherent cells have to be detached from the plate (scrapping or trypsinizing), centrifuged and resuspended. The medium depends on your protocol – if you want to permeabilize the cells, you need a mitochondrial respiration medium (http://www.oroboros.at/index.php?id=protocols_miro6), in culture medium the Ca 2+ concentration is too high for mitochondria.== Appropriate cell number 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. Just to give an estimate (values are corrected for background):maximum flux up to 100 to 150 pmol/sec/ml minimum fluxes at 5 pmol/sec/ml == Primary hepatocytes or fibroblasts ==Primary isolated hepatocytes or fibroblasts are quite large, therefore, 0.1 to 0.3 million cells/ml can be applied.== HUVEC ==Although HUVEC are primary cells, they are comparable to cultivated cells, 1.0 million cells/ml is recommended.thymocytes and lymphocytes:A cell number as suggested for cultivated cells, 1.0 million cells/ml, is appropriate.cultivated cells:A lot of cultivated cells are comparable in their size and mitochondrial density.A sample concentration of approximately 1.0 million cells/ml is appropriate for many cultured cell lines like different cancer cell lines or immortalized cell lines.