Winmdi software

The surface marker expression of isolated HWJ-MSCs was investigated using flow-cytometry according to the described procedure.^{20 (link)} In brief, 1×10⁵ cells/mL (passage three) were washed with PBS, fixed and permeabilized with 0.2% Triton X-100 and incubated with normal goat serum diluted in PBS (1:9) for 15 min at 4°C for blocking non-specific binding of the primary antibodies. The cells were incubated with the specific antibody conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (Santa Cruz) at concentrations recommended by the respective manufacturer’s for 1 h and then analyzed by a flow cytometer. The antibodies conjugated by FITC were: anti-CD44, anti-CD34 (Chemicon, USA) and anti-CD45 (eBioscience, USA), and those conjugated by PE were anti-CD73 (Becton Dickinson [BD], USA), anti- CD90 (Dako, Denmark) and anti-CD105 (R&D, USA). Following incubation, the cells were washed with 2% FBS in PBS and run through a BD FACSCalibur (USA) flow cytometer. The control group was stained with isotype-matched antibodies (FITC- and PE-conjugated mouse IgG monoclonal isotype standards), which were confirmed by positive fluorescence of the limbal samples. Typically for each tube, 10 000 events were collected and the data analysis was performed using WinMDI software (BD Biosciences, USA).

Viable hUCMs (1×10⁵ cells) were fixed with 4% paraformaldehyde for 15 minutes, washed by washing buffer, incubated with 200 μl of 10% goat serum for 15 minutes, and washed again by washing buffer, after which mouse anti-human antibodies conjugated with phycoerythrin (PE) against CD34, CD45, CD73, and CD90 were added. The mixture was incubated for one hour at 4˚C (19 (link)). The samples were washed by washing buffer and assessed by flow cytometry (BD FACS Calibur, USA). For each antibody, at least 10000 events were recorded per sample and data were analyzed by WinMDI software (BD Biosciences, CA).

The control and treated splenocytes were collected, washed with ice-cold PBS and fixed with 75% ethanol at 4°C for 16 hr. After fixation, cells were washed twice with ice-cold PBS and incubated in 1 mL of a solution containing 0.1% Triton X-100, RNase A (39.5 µg/mL) and PI (20 µg/mL) for 30 min. The fluorescence emitted from the PI-DNA complex was measured at 488 nm excitation/600 nm emission using a FACScan instrument (Becton Dickinson, San Jose, CA, USA), and the percentage of cells below the G1 peak (subG0/G1 fraction) was analyzed using the WinMDI software (Becton Dickson, San Jose, CA, USA).