Garcia-Souza 2018 Life Sciences Meeting 2018 Innsbruck AT
|A respirometric cell viability test for peripheral-blood mononuclear cells and platelets.|
Mitochondrial (mt) respiration of blood cells represents a powerful diagnostic model for evaluation of mt-function in health and disease. However, standard operating procedures have to be established. We developed a substrate-uncoupler-inhibitor titration protocol to measure respiratory control in intact cells and cell viability in an integrated assay: the coupling control and cell viability protocol (CCVP) using mitochondrial respiration medium MiR06 and cell culture media RPMI and M199.
Respiration was determined in isolated peripheral blood mononuclear cells (PBMC) and platelets from healthy volunteers by high-resolution respirometry. ROUTINE respiration of intact cells (ce) was measured before and after addition of pyruvate. Oligomycin (0.05 – 2.5 µM, inhibitor of F-ATPase) was added to obtain LEAK respiration. Electron transfer-capacity (E) was stimulated by titration of CCCP (uncoupler), followed by glucose addition (Crabtree effect). Malate was added to stimulate respiration of cells without intact plasma membrane (dead cells, dce). Rotenone inhibits intact cells to the level of residual oxygen consumption (Rox), after which succinate stimulates respiration of dce. The entire cell population was permeabilized by titration of digitonin, providing a reference level of succinate respiration (permeabilized cells, pce). Cytochrome c was added to test the intactness of the mt-outer membrane (mtOM). CIII was inhibited by antimycin A, to measure CIV activity by addition of ascorbate and TMPD and later inhibition by azide.
ROUTINE respiration in MiR06 was higher in PBMCs but lower in platelets compared to cell culture media. Pyruvate stimulated respiration (p<0.02) in both cell types and respiration was higher in MiR06 than in cell culture media containing glucose after uncoupling. 0.01 µM oligomycin inhibited LEAK respiration as much as 2.5 µM, without or only slight inhibitory effect on E in PBMCs and platelets, respectively. In PBMCs, the CCVP viability test, 1-(dce-Rox)/(pce-Rox), resulted in 94% viability which correlated with 96% obtained with acridine orange/propidium iodide staining.
The respirometric CCVP viability test was equally applied to platelets. Additionally, the CCVP protocol yields a measure of the integrity of the mtOM and CIV activity. Taken together, our results suggest that the CCVP offers a standard operating procedure for mitochondrial respiratory studies in blood cells.
- Garcia-Souza LF(1,2), Cizmarova B(1,3), Volani C(4), Sumbalova Z(1), Doerrier C(5), Gnaiger E(1,5)
- Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
- Inst Sport Science, Univ Innsbruck, Austria
- Dept Medical Clinical Biochem, Fac Medicine, Pavol Jozef Šafárik Univ Košice, Slovakia
- Dept Internal Medicine II, Medical Univ Innsbruck, Austria
- Oroboros Instruments, Innsbruck, Austria. - firstname.lastname@example.org
Labels: MiParea: Respiration, Instruments;methods
Organism: Human Tissue;cell: Blood cells, Platelet Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET Pathway: S HRR: Oxygraph-2k Event: Poster MitoEAGLE