Cytoflex v2 b4 r2 flow cytometer

Flow cytometry experiments were performed on a CytoFLEX V2-B4-R2 Flow Cytometer (eight detectors, three lasers) (Beckman-Coulter, Germany). Antibodies used for surface staining are listed in ESM Table 1. For measuring intracellular thiol levels, ThiolTracker dye (T10095, Thermo Fisher Switzerland), was used according to the manufacturer’s directions. Cells were washed and subsequently analysed.

Flow cytometry experiments were performed on a CytoFLEX V2-B4-R2 Flow Cytometer (8 Detectors, 3 Lasers) (Beckman Coultier, Germany). In short, cells were harvested as described above and subsequently stained with the appropriate antibodies and dyes. For flow cytometry experiments, BMDMs or peritoneal lavage was harvested. F4/80 APC-Cy7 (Cat nr. 123113, Biolegend, UK) and CD11b PE-Cy7 (Cat nr. 101215, Biolegend, UK) positive cells were considered macrophages.
For measuring intracellular thiol levels, Thioltracker dye (T10095, Thermo Fisher Switzerland), was used according to manufacturer’s directions.

The differentiated cells in T12.5 flask were dissociated into single cells using 0.05% trypsin supplemented with 0.1% EDTA. The cells were washed, resuspended in a FACS washing buffer (PBS with 5% BSA and 2.5 mM EDTA) and then stained with the desired antibodies. The antibodies used in our study were: PE Mouse Anti-Human CD31 (1:25, BD), FITC Mouse Anti-Human CD34 (1:25, BD), APC Mouse Anti-Human CD34 (1:25, BD), APC Mouse Anti-Human CD43 (1:25, BD), APC Mouse Anti-Human CD45 (1:25, BD), FITC-conjugated mouse IgG2a (1:25, BD), APC-conjugated mouse IgG1 (1:25, BD) and PE-conjugated mouse IgG1κ (1:25, BD) were used as isotype-matched negative controls. Flow cytometry was performed on Calibur flow cytometer (BD) or CytoFLEX V2-B4-R2 Flow Cytometer (BECKMAN COULTER), and the data was analyzed using FlowJo software, version 10.0.7.