Fvs780 dye

HFF were analysed with the following anti-human monoclonal antibodies: allophycocyanin (APC)—conjugated mouse anti-human CD10 (clone eBioCB-CALLA, eBioscience, Thermo Fisher Scientific) in combination with phycoerythrin (PE)—conjugated mouse anti-human C5a receptor-like 2 (GPR77 or C5L2, clone 1D9-M12, BioLegend, San Diego, CA, USA) and, in parallel, with APC-IgG2b, k (eBioscience) and PE-IgG2a, k (BioLegend) isotype controls. To quantify ALDH1 activity, the ALDEFLUOR Kit (STEMCELL Technologies, Vancouver, BC, Canada) was used according to the manufacturer’s protocols. All analyses were performed on viable cells, stained with FVS780 dye (BD Biosciences, Milan, Italy).
Flow cytometry acquisition of stained cells was conducted using a LSR II flow cytometer (BD Biosciences) and data were analysed using Kaluza Analysis software Ver. 1.3 (Beckman Coulter, Brea, CA, USA).

Samples were transferred to a 96-well Nunc conical well plate (Sigma-Aldrich Corp.) and spun at 450g at 4°C for 5 minutes. Cells were resuspended in 100 μL flow buffer (1× PBS with 1% BSA) with 5 ng/μL FC block (purified anti-CD32 clone D34-485, BD Biosciences, Reading, UK) and incubated at 4°C for 15 minutes. Staining was performed in the dark for 20 minutes at 4°C in flow buffer. The list of titrated surface markers for these experiments includes: CD3 clone 1F4 FITC (5 ng/μL), CD45R clone HIS24²⁰ PE (4 ng/μL), CD8 clone OX-8 PerCP (2 ng/μL), and CD4 clone OX-35 PE-Cy7 (2 ng/μL). Live-dead discrimination was performed using FVS780 in the APC-Cy7 channel. All stains including the FVS780 dye were obtained from BD Biosciences. Unstained cell controls were used for gaiting analyses to distinguish positively from negatively staining cell populations. Compensation was performed using single color controls prepared from BD Comp Beads (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometric analysis was carried out with a FACS Canto II equipped with 488 and 647 nm excitation lasers. Data analysis was performed using FlowJo v10.1 software (FlowJo LLC, Ashland, OR, USA). Data presented represents results from three independent experiments. Population percentages and statistical results are presented for samples where >10,000 events were captured in the inflammatory gate.