For cell surface marker analysis, cells were stained with antibodies in PBS containing 0.1% (wt/vol) BSA and 0.1% NaN3. The following mAbs were used in the present study: anti-CD11b (M1/70; eBiosciences, Ben Lomond, CA, USA), anti-Gr1 (RB6-8C5; eBiosciences), anti-CD45 (TU116; BD Biosciences, San Diego, CA, USA), anti-GR (EPR4595; Abcam, Cambridge, MA, USA), anti-HIF1α (IC1935P; R&D system, Minneapolis, MN, USA), anti-human HLADR (L243; Biolegend, San Diego, CA, USA), anti-human CD33 (P67.0; Biolegend) and anti-human CD11b (ICRF44; Biolegend).
Intracellular staining was analysed by FCM as described.18 (link) Cells isolated from different organs were re-stimulated with LPS (L2630; Sigma, St Louis, MO, USA) for CD11b+Gr1+ cell analysis for 5 h, and GolgiStop (554724; BD Bioscience) was added for the last 2 h. After surface staining and washing, the cells were immediately fixed with cytofix/cytoperm solution (554714; BD Biosciences) and were stained with anti-TNFα (MP6-XT22; eBiosciences) and anti-IL-10 (JES5-16E3; eBiosciences). For transcriptional factor analysis, after surface staining, the cells were fixed with fixation/permeabilization buffer (00-5523; eBioscience) and stained with anti-GR and anti-HIF1α. FCM data were acquired on a FACSCalibur (Becton Dickinson, San Diego, CA, USA), and data were analysed with FlowJo (TreeStar, San Carlos, CA, USA).
Intracellular staining was analysed by FCM as described.18 (link) Cells isolated from different organs were re-stimulated with LPS (L2630; Sigma, St Louis, MO, USA) for CD11b+Gr1+ cell analysis for 5 h, and GolgiStop (554724; BD Bioscience) was added for the last 2 h. After surface staining and washing, the cells were immediately fixed with cytofix/cytoperm solution (554714; BD Biosciences) and were stained with anti-TNFα (MP6-XT22; eBiosciences) and anti-IL-10 (JES5-16E3; eBiosciences). For transcriptional factor analysis, after surface staining, the cells were fixed with fixation/permeabilization buffer (00-5523; eBioscience) and stained with anti-GR and anti-HIF1α. FCM data were acquired on a FACSCalibur (Becton Dickinson, San Diego, CA, USA), and data were analysed with FlowJo (TreeStar, San Carlos, CA, USA).
