Buv395 conjugated anti cd45

Following humane euthanasia, wounds were harvested using 6 mm punch biopsy to collect 2 mm wound margins for analysis. Three pools of 2 wounds per animal were chopped and digested with Liberase TL enzyme cocktail [0.35 mg/mL Liberase TL (Roche), 3 mg/mL Collagenase D (Roche) and 0.1 mg/mL DNase I (Roche)] for 2 h at 37°C.25 (link) After incubation, the samples were filtered through a 70-µm strainer to remove undigested debris and to yield single-cell suspensions. Approximately 1×10⁶ cells were incubated with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences) and with a Zombie Aqua™ viability dye (BioLegend) for 15 min. After washing with FACS buffer (0.5% bovine serum albumin (BSA); 2 mM EDTA in PBS), surface receptors were stained with BUV395-conjugated anti-CD45 (BD), BV786-conjugated anti-CD11b (BD), phycoerythrin (PE)-conjugated anti-Ly6G (eBioscience) and PE/Cy5-conjugated anti-F4/80 (eBioscience) for 30 min. Next, cells were washed and fixed with 0.37% formaldehyde in PBS. Cells were analyzed on a BD LSR Fortessa X20 with DIVA software. Results were further analyzed with the FlowJo software (BD Biosciences).