Cd24 fitc

50 000 viable B16-F10 cells (trypan blue exclusion test) dissociated from one-week-old tumorspheres or from trypsinized adherent monolayers were incubated for 30 minutes at 4°C in the dark with 0.5 µg of rat anti-mouse monoclonal CD133-APC, CD44-FITC or CD24-PE antibodies (eBiosciences, Paris, France) in 100 µL of a buffer solution consisting in PBS containing 3% bovine serum albumin (Sigma). Immunostaining was also performed for HT-29, MCF-7 and MDA-MB-231 cell lines using mouse anti-human monoclonal CD133-APC, CD44-PE, CD44-APC or CD24-FITC antibodies (Miltenyi Biotec, Paris, France) according to the manufacturer’s instructions. Cells were then washed and analyzed with an Accuri C6 flow cytometer (BD biosciences, USA).

The cells were detached, dissociated into single cells using dispase (Sigma-Aldrich), and resuspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline PBS, 1% BSA; 5 × 10⁵ cells/100 μl FACS buffer). For cell-surface staining the cells were stained with primary antibodies CD73-PE (130-095-182, Miltenyi), CD105-FITC (130-094-926, Miltenyi), CD90- PE-VIO770 (130-099-295, Miltenyi), CD45-PE (130-098-141, Miltenyi), CD34-FITC (130-098-139, Miltenyi), CD24-FITC (130-098-861, Miltenyi), SSEA4-PE (FAB1435P, R&D), and TRA1-60-PE (09-0009, Stemgent) for 1 hour at 4 °C. For intracellular FACS staining, i.e. OCT4-A (130-105-606, Miltenyi) and OCT4-B (IC1759P, R&D), the cells were washed once in FACS buffer, fixed for 10 min in 0.01% paraformaldehyde (PFA), washed twice with PBS, resuspended in permeabilization buffer (PBS, 1% Triton) and stained as describe above. Cells were then washed twice with PBS, resuspended in FACS buffer and analysed by FACScalibur flow cytometry (CyAn ADP, Beckman Coulter). Collected data were analyzed with the Flowjo v.10 software package (Treestar, Ashland, OR, USA). Negative control was immunoglobulin G (IgG) primary antibody-specific isotypes.

BD Stemflow™ Human MSC Analysis Kit (BD biosciences, California, USA), CD24-FITC, and CD146-PE (both bought from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were used on sorted CD105+ cells and were acquired using FACS Calibur for enumeration of CD24, CD146, CD73, CD90, CD105, CD11b, CD19, CD34, CD45, and HLA-DR markers. All antibody staining was prepared according to manufacturer protocol. Each sample was run with proper isotype control.