Epics xl 4 color cytometer

Cell proliferation was measured by incorporation of fluorescence-conjugated EdU to the newly synthesized DNA, according to the manufacturer’s protocol (Click-iT EdU Alexa Fluor; Invitrogen). Briefly, ovarian cancer cells were plated at a density of 5 × 10⁴ cells in a 6-well dish and transfected with siRNAs. Forty-eight hours after transfection, tumor cells were treated with 10 mM EdU for 2 hr, washed three times with PBS, detached with 0.25% EDTA, fixed with 4% paraformaldehyde for 15 min, immunostained with anti-EdU-FITC (1:200), and analyzed using flow cytometry (EPICS XL 4-Color Cytometer; Beckman Coulter).

Fifty thousand cells IG10 or ID8 were plated in 6-well plates and grown in serum-free media (SFM) for 24 h. Mouse platelets (20 × 10⁶ per well) isolated as described above were added to the cells. After 48 h of incubation, cell proliferation was analyzed using the BrdU labeling and Detection Kit (Roche, Indianapolis, USA) according to the manufacturer’s instructions. Briefly, cells were pulse-labeled with 10 μM BrdU for 90 min, washed three times with PBS, detached with 0.25% Trypsin-EDTA, fixed with 70% ethanol fixative for 20 min, immunostained with mouse anti-BrdU primary antibody and anti-mouse Ig-fluorescein secondary antibody, and finally analyzed using flow cytometry (EPICS XL 4-Color Cytometer; Beckman Coulter, Brea, USA). The assay was performed for 3 times.