Pe cyanine7 anti mouse f4 80 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).

Primary BMDMs were induced under specific conditions and labeled with the following antibodies for identification of M1 or M2 macrophages: PE/Cyanine7 anti-mouse F4/80 Antibody (Biolegend, 123113) PE/Cyanine5 anti-mouse CD86 Antibody (Biolegend, 105015), FITC anti-mouse CD206 (MMR) Antibody (Biolegend, 141703). Related isotypes for control are PE/Cyanine7 Rat IgG2a, κ Isotype Ctrl Antibody (Biolegend, 400521), PE/Cyanine5 Rat IgG2a, κ Isotype Ctrl Antibody (Biolegend, 400509), FITC Rat IgG2a, κ Isotype Ctrl Antibody (Biolegend, 400505). Cells were suspended and incubated with antibodies for 30 min, and analyzed via Guava Easycyte 12HT (Luminex).