2.6. Flow Cytometry

The purity of sorted cells was monitored by flow cytometry. A total of 1 × 10⁵ cells were incubated with primary antibodies on ice for 30 min in PBS containing 2 mM EDTA and 2% BSA. The following antibodies were used at specific dilutions: anti-α7-Integrin APC 1:500 (Ablab, Vancouver, Canada, #67001005), anti-Sca1 FITC 1:50 (BD Pharmingen, Milan, Italy, #557405), anti-CD31 PE 1:100 (BD Pharmingen #553373) and anti-CD45 eFluor 450 1:50 (eBioscience, Thermo Fisher Scientific, Monza, Italy, #48-0451-80).

Cell suspensions were washed with 2 mL of 2 mM EDTA and 2% BSA in 1X PBS and centrifuged at 4 °C for 10 min at 700× g. Unstained samples were prepared as control. Pellets were then resuspended in 100 µL of 2 mM EDTA and 2% BSA in 1X PBS and run to a flow cytometer. Approximately 10,000 events per sample were acquired with a CytoFLEX S (Beckman Coulter, Milan, Italy) instrument equipped with three lasers (488 nm, 405 nm and 638 nm) and 13 detectors. Quality control of the cytometer was assessed daily using CytoFLEX Daily QC Fluorospheres (Beckman Coulter #{"type":"entrez-nucleotide","attrs":{"text":"B53230","term_id":"2607564"}}B53230). Data were collected by CytExpert 2.2 version (Beckman Coulter) software. If needed, a compensation matrix was calculated using a VersaComp Antibody Capture Kit (Beckman Coulter #{"type":"entrez-nucleotide","attrs":{"text":"B22804","term_id":"2508479"}}B22804) according to the manufacturer’s instructions. FCS files were analyzed using CytExpert version 2.2 software.