Transcription factor fixation permeabilization buffer

After lysis of erythrocytes in lithium heparin-anticoagulated blood using Pharm Lyse solution (Becton Dickinson, San Jose, CA), surface staining was performed using fluorochrome-conjugated antibodies (CD15-FITC (Clone H198), CD3-PECγ7 (Clone HIT3a), CD14-APC (Clone M5E2) (BioLegend Cat# 301807, RRID:AB_314189) (all obtained from BioLegend, San Diego, USA) for neutrophils, lymphocytes, and monocytes, respectively. After fixation and permeabilization with Transcription Factor Fixation/Permeabilization buffer (eBioscience Inc., San Diego, USA), PE-conjugated anti-HIF-1α and corresponding isotype control (Clone 241812, RD Systems, Minneapolis, USA)were used to stain intracellular HIF-1α and correct for aspecific binding. Leukocytes were fixed and stored overnight in PBS containing 1% bovine serum albumin and 1% paraformaldehyde. The data were acquired with Cytomics FC500 (Beckman Coulter, Brea, CA, USA) and analyzed with Kaluza software (Beckman Coulter). Cell subtypes were identified by surface staining and sideward scatter. Mean PE fluorescence, after subtraction of isotype-fluorescence, was used as a measure of HIF-1α expression. The gating strategy used is depicted in Fig. S3.