Pe anti mouse cd11b antibody

After the isolation of stromal vascular fraction of PVAT, cells were suspended in PBS containing 5% FBS. Zombie NIR Fixable Viability Kit (BioLegend) was used to label dead cells according to the manufacturer’s instructions. Nonspecific binding of the antibodies to Fc receptors was blocked by using Mouse Fc receptor-blocking agent (BioLegend). Cells were incubated at room temperature 30 mins with FITC anti-mouse CD45 Antibody (BioLegend, #103107, 1:50), PE/Cyanine7 anti-mouse F4/80 antibody (BioLegend, #123113, 1:50), PE anti-mouse CD11b antibody (BioLegend, #101207, 1:50), APC anti-mouse CD206 antibody (BioLegend, #141707, 1:50)‚ and Brilliant Violet 421 anti-mouse CD11c antibody (BioLegend, #117329, 1:50), then centrifuged and fixed with PBS containing 2% PFA. Cells were analyzed using a FACSMelody cell sorter (BD Biosciences). For the analysis of flow cytometry data, CD45⁺, CD11b⁺, and F4/80⁺ cells were defined as macrophages, which could be further differentiated into classically (M1; defined as CD11c⁺, CD206⁻) and alternatively activated (M2; defined as CD11c⁻, CD206⁺) macrophages^{77 (link)}. Data analyses were performed using FlowJo software (Tree Star).

AT-infiltrating stromal cells were stained with FITC anti-mouse CD3ε antibody, PE anti-mouse CD4 antibody, PE/Cy7 anti-mouse CD25 antibody, APC anti-mouse CD8a antibody, FITC anti-mouse CD127 antibody, PerCP/Cy5.5 anti-mouse CD4 antibody, APC anti-mouse CD25 antibody (these antibodies were purchased from Biolegend, San Diego, CA, USA), PE anti-mouse/Foxp3 antibody (Invitrogen, Carlsbad, CA, USA), PE anti-mouse CD11b antibody (Biolegend), PE/Cy7 anti-mouse Ly-6C antibody (Biolegend, San Diego, CA, USA) and APC anti-toll-like receptor 4 (TLR4) antibody (Invitrogen, Waltham, MA, USA), and analyzed using an Accuri C6 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).