Retone antimycin a

Mitochondrial activity of freshly isolated progenitor cells, stem cells and macrophages, neutrophils and B cells was measured using a seahorse metabolic influx assay according to the manufacturer’s instructions (Agilent Technologies). Optimisation runs identified the ideal number of cells per well for the respective cell types using the 96-well plate assay format (10,000–25,000 HSPC and HSC/well, 25,000 macrophages/well, 50,000 neutrophils/well, 100,000 B cells/well). Cells were re-suspended in serum-free seahorse XF DMEM medium (pH 7.4 supplemented with 25 mM glucose and 1 mM pyruvate) and plated onto a 96-well plate pre-coated for 1 h with Cell-Tak (22.4 μg/ml, Corning). Plates were immediately analysed using a seahorse Bioscience XFe96 extracellular flux analyser (Agilent Technologies). The oxygen consumption rate (OCR) was measured at 37 °C under basal conditions and in response to 1 μM oligomycin, followed by 2 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP) and 1 μM retone/antimycin A (all from Agilent Technologies). The OCR from cells was normalised against the total protein concentration of cells measured by a DC protein assay (Bio-Rad) according to the manufacturer’s instructions. Every seahorse assay was run with the following instrument settings: 7 min mix time and 4 min read time.

The mitochondrial activity of freshly isolated hematopoietic stem and progenitors was measured using a seahorse metabolic influx assay (AgilentTechnologies, USA, Agilent Seahorse Wave Desktop Version 2.2.1.5 was used). Fifteen thousand cells per well were resuspended in a serum-free seahorse XF DMEM medium (pH7.4 supplemented with 25 mM glucose and 1 mM pyruvate) and plated onto a 96-well plate pre-coated with Cell-Tak (22.4 μg/ml, Corning, USA). The plate was analysed immediately according to the manufacturer’s instructions using a seahorse Bioscience XFe96 extracellular flux analyser (AgilentTechnologies, USA). The oxygen consumption rate (OCR) was measured at 37 °C under basal conditions and in response to 1 μM oligomycin, 2 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP) and 1 μM retone/antimycin A (AgilentTechnologies, USA). The seahorse assay was run in triplicate with the following assay conditions: 7 min mix time and 4 min read time. The OCR from cells were normalised against total protein measured by a DC protein assay (Bio-Rad, USA).