Bv510 conjugated anti ly6c

Hydrogels were explanted from the subcutaneous space, weighed, and placed in cold PBS containing calcium and magnesium. Hydrogels were washed for 30 min on a shaker to remove nonadherent cells. Adherent cells were isolated by digesting hydrogels with collagenase type 1A (1 mg/ml) at 37°C for 30 min and further disaggregated with a cell strainer to ensure a single-cell suspension. Single-cell suspensions were stained for flow cytometry analysis in 3% fetal bovine serum/PBS according to standard procedures and analyzed on a FACSAria IIIu flow cytometer (BD Biosciences). The following antibodies were used for immunophenotyping: BV421- or BV510-conjugated anti-CD11b (BioLegend), anaphase-promoting complex (APC)– or BV510-conjugated anti-Ly6C (BioLegend), PerCP-Cy5.5–conjugated anti-CD11c (BioLegend), APC-Cy7–conjugated anti-Ly6G (BioLegend), BV711-conjugated anti-CD64 (BioLegend), phycoerythrin (PE)–conjugated anti-MerTK (BioLegend), PE-Cy7– or BV605-conjugated anti-CD206 (BioLegend), and fluorescein isothiocyanate (FITC)–conjugated anti-CD86 (BioLegend). Staining using BV dyes was performed in the presence of Brilliant Stain Buffer (BD Biosciences). Positivity was determined by gating on fluorescence minus one control. Absolute quantification of cell numbers was performed by adding 25 μl of AccuCheck counting beads to flow cytometry samples (Thermo Fisher Scientific).