Anti mouse cd206 bv421

After digestion, the single-cell suspension was generated and filtered through a 40 µm preseparation column, centrifuged at 400×g for 5 mins, and resuspended in PBS. A Zombie Aqua™ Fixable Viability Kit (Biolegend, 423101) was used to identify live cells for 20 min in the dark according to the manufacturer’s instructions. After that, the suspension was centrifuged again at 400×g for 5 mins and resuspended in stain buffer (BD Biosciences, 554657). Purified anti-mouse CD16/32 (Biolegend, 101301) was used to block Fc of the cells on ice for 5 min, and then the cells were stained at 4°C for 30 min with the following antibodies: anti-mouse CD45-APC/750 (Biolegend, 103154), anti-mouse CD11b-FITC (Biolegend, 101206), anti-mouse Ly6G-PE/Cy7 (Biolegend, 127618), anti-mouse F4/80-APC (Biolegend, 123116), anti-mouse Ly6C-PE (Biolegend, 128007), anti-mouse I-A/I-E-BV605 (Biolegend, 107639), anti-mouse CD206-BV421 (Biolegend, 141717), or anti-mouse C-C motif chemokine receptor 2 (CCR2)-BV421 (Biolegend, 150605). Next, stain buffer was added, and the cells were washed twice. Flow cytometry analysis was performed on the second day, and the data were analyzed with FlowJo software.

BMC were isolated from normal mice (on day 0), and cultured with recombinant mouse GM-CSF in complete RPMI 1640 medium for 4 days (on day 4) for defferentiation of MDSC. MDSC induced in vitro from BMC were collected and blocked with Anti-Mouse CD16/32 antibody (Biolegend, USA) for 15 min, and then stained with fluorescence-labeled antibody Anti-Mouse CD11b-FITC (BD Biosciences, USA) or Anti-Mouse CD11b-BV421 (BD Biosciences, USA), in the dark at 4°C for 30 min, followed adding Fixation and Permeabilization buffer (BD Biosciences, USA, 250 μl/tube), at 4°C for 20 min. Cells were washed with 1 ml of 1×Perm/Wash™ buffer (BD Biosciences, USA), and finally, Anti-Mouse CD206-AF647 (BD Biosciences, USA) or Anti-Mouse CD206-BV421 (Biolegend, USA) was added and incubated at 4°C for 30 min in the dark and detected by BD FACSVerse flow cytometer and quantified with FlowJo 7.6 software.

The expression of CD11b, CD206, CD11c, and F4/80 in BMSC-derived macrophages were analyzed using the FACS Calibur (Becton Dickinson, United States) and the CellQuest software (Becton Dickinson). The adherent cells were washed with PBS and collected using Accutase (Nacalai Tesque, Kyoto, Japan) and resuspended in 50 μL staining buffer (BD Pharmingen, Franklin Lakes, NJ, United States). The harvested cells were blocked with 2 μL Human Trustain FcX (Fc receptor Blocking Solution; BioLegend) for 10 min at room temperature, and stained with 2 μL antibodies in the dark for 30 min at 4°C. FITC anti- mouse CD11b, FITC anti-human CD11b, Alexa Fluor^®488 anti-human CD11b, BV421 anti-mouse CD206, PE anti-mouse F4/80 and APC anti-mouse CD11c (BioLegend).