Hla 1

For cytofluorimetric analysis adherent cells were trypsinized, then washed twice in PBS, incubated with 5 μl of fluorochrome-conjugated antibodies for 30 min at 4 °C (5 × 10⁵cells/FACS tube), fixed with 1% formaldehyde solutions for 5 min at 4 °C, centrifuged for 5 min at 1600 rpm and washed once with 1 ml of PBS for 5 min at 1600 rpm. CD133/2 (293C3) (Miltenyi Biotec, Bergisch Gladbach, Germany), HLA-I, NGF-R, ICAM-1, CD20, CXCR4, CD10, c-Kit antibodies (all from BD Biosciences, Franklin Lakes, NJ, USA) conjugated with different fluorescent dyes were used; the unconjugated antibodies Nestin (Novus Biologicals, Minneapolis, USA) and Melan-A/MART-1 (Santa Cruz Biotechnology, Dallas, TX, USA) were used in combination of the goat-anti-mouse IgG-FITC (BD Biosciences) as secondary antibody and used after cell permeabilization. For apoptosis analysis cells were trypsinized, washed in PBS, fixed with 70% ethanol for 45 min at 4 °C, washed in PBS and stained with propidium iodide (50 μg/ml diluted in PBS) and RNAase (250 μg/ml), then stored for at least 3 h at 4 °C before analysis. Flow cytometer analysis was performed by BD FACScan™ System using CellQuest Pro software on a minimum of 5000 events for each sample.

For the characterization of epithelial-like cells compared with HERS/ERM, fluorescence-activated cell sorting (FACS) was performed. The cells were detached and washed with DPBS supplemented with 2% FBS and then fixed with 4% paraformaldehyde at RT for 10 min. After washing with DPBS, 10,000 cells were incubated with fluorescently conjugated antibodies for 20 min at 4 °C. The following antibodies were used: FITC-conjugated mouse anti-human CD31, HLA-DR, HLA-I, PE-conjugated mouse anti-human CD10, CD29, and APC-conjugated mouse anti-human CD45 (all from BD Pharmingen, San Diego, CA, USA). The fluorescence intensity was measured by a FACS Calibur, and data were analyzed with FlowJo software (Tree Star, San Carlos, CA, USA).