Mitokit cii

Mitochondrial respiration was assessed with the O2k–Oxygraph (Oroboros Instruments GmbH Innsbruck, Austria) and a buffer (MiR05) containing: 0.5 mM EGTA, 3 mM MgCl₂, 60 mM K–lactobionate, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM sucrose and 1 g/L bovine serum albumin [26 ]. Platelets were suspended in the 2 mL glass chamber at a concentration of 200 × 10⁶ cells/mL at 37 °C. All chemicals were obtained from Sigma–Aldrich. The cell–permeable succinate prodrug NV118 was kindly provided by Abliva AB (Lund, Sweden). It is also available via Oroboros Instruments in the MitoKit-CII (MitoKit-CII/">https://www.oroboros.at/index.php/product/MitoKit-CII/ (accessed on 22 March 2021)).

All chemicals were obtained from Sigma-Aldrich (Saint Louis, MO, USA). The cell-permeable succinate prodrug was kindly provided by Abliva AB (Lund, Sweden) [18 (link)], and is also available via Oroboros Instruments in the MitoKit-CII (MitoKit-CII/">https://www.oroboros.at/index.php/product/MitoKit-CII/).
Platelets were isolated from venous blood from healthy volunteers both men and women, aged between 27–32 (with one exception, a female aged 66), drawn in K₂EDTA tubes according to a previously described protocol [15 (link)]. In brief, platelets were subjected to a sequence of differential centrifugations; the first at 500× g for 10 min and the second at 4600× g for 5–10 min.

All chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA). NV118 (cell-permeable succinate prodrug) was generously donated by Abliva AB (Lund, Sweden). This compound is also available as the MitoKit-CII from Oroboros Instruments (MitoKit-CII/">https://www.oroboros.at/index.php/product/MitoKit-CII/) (accessed on 1 September 2021).
Human blood cells (platelets and PBMCs) isolation was carried out from venous blood samples provided by a group of healthy volunteers, both men and women. The blood was drawn in K₂EDTA tubes. Isolation was achieved by means of differential centrifugation, as previously described by Sjövall et al. [24 (link)]. Oxygen consumption was normalized to the number of cells.