Living cells

high-resolution terminology - matching measurements at high-resolution

Living cells

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 

MitoPedia topics: "Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property. Respiratory state"Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property. 

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

The lab of Gregory Brewer developed techniques for high-resolution respirometry with the OROBOROS-O2k of neuronal cells attached to a substrate:

• Jones TT, Brewer GJ (2009) Critical age-related loss of cofactors of neuron cytochrome c oxidase reversed by estrogen. Exp Neurology 215: 212-219
• Jones TT, Brewer GJ (2010) Age-related deficiencies in Complex I endogenous substrate availability and reserve capacity of Complex IV in cortical neuron electron transport. Biochim Biophys Acta Bioenergetics 1797: 167-176.

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 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-C2061.

Fibroblasts, HUVEC, thymocytes, lymphocytes

1.0 million cells/ml is recommended for many cultured cells, including different cancer or immortalized cell lines.

Hepatocytes

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