Cell quest acquisition and analysis software

Annexin V was measured using a kit obtained from Molecular Probes (Invitrogen). Cells were resuspended in annexin binding buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) containing Alexa Fluor 568–conjugated annexin V and incubated in the dark for 15 min. Samples were put on ice and immediately analyzed. The extent of staining was monitored by fluorescence-activated cell sorting (FACS) using a FACSCalibur (Becton-Dickinson) and CellQuest acquisition and analysis software (Becton-Dickinson) immediately after staining. On several occasions, annexin V staining was also monitored in adherent cells using a Nikon Eclipse TE300 fluorescence microscope and quantified using OpenLab image analysis software (ImproVision).

U-87 MG cells were incubated with 80 µg/mL and 100 µg/ mL Bacoside A for 24 h before the cells were harvested. The treated and untreated cells were washed twice in 1× PBS (4,000 rpm, 10 min, 25℃), and the cell pellet obtained was resuspended in Annexin V binding buffer at room temperature to a concentration of 1×10⁶ cells/mL. To the resuspended solution, 5 µL of Annexin V-FITC and 5 µL of PI were added. The resulting mixtures were gently mixed and incubated for 15 min at room temperature in dark. An additional 400 µL of binding buffer was added to each tube and flow cytometric evaluation was conducted immediately on a FACSCalibur™ flow cytometer using Cell Quest acquisition and analysis software (Becton Dickinson).

The RGCs’ purity was evaluated using a flow cytometer (FACSCaliber; Becton Dickinson). As most of the Thy-1 antigens on the surface of the RGCs were bound to the corresponding antibody during the separation and purification process, we adopted another RGC-specific marker, Brn3a, as a surrogate marker for the RGCs. The steps are summarized as follows: The RGCs that had been separated and purified using the three methods were separately fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Then, the cells were centrifuged and resuspended in PBS containing 0.4% Triton X-100 (Sigma), and 3% BSA was added to block non-specific binding of the antibodies. Each sample was subsequently centrifuged and incubated with a rabbit anti-rat Brn3a antibody (1:50, Abcam) for 60 min at 4 °C. After washing with PBS, the cells were exposed to fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit immunoglobulin G (IgG; 1:400, Life Technologies) in 1.0 ml of PBS for 1 h at RT. After washing twice with PBS, the cells were excited with a 488-nm laser and collected in the FITC (515–545 nm) channels. Cell Quest Acquisition and Analysis software (Becton Dickinson) was used to quantify the intensities of the fluorescent signals and to construct dot density plots.

Transfected OCI-Ly7 cells (1×10³ cells/well) were seeded into a 96-well plate and cultured for 48 h at 37°C. For cell cycle analysis, transfected OCI-Ly7 cells were collected and fixed with 70% ethanol at 4°C overnight. After washing with PBS, the transfected OCI-Ly7 cells were incubated with 1 mg/ml RNase A for 20 min at 37°C, followed by staining with propidium iodide (PI) and 1% Triton X-100 for 20 min at 4°C. The experimental data were collected using a flow cytometer (BD FACScan; Becton, Dickinson and Company). For apoptosis analysis, cells were washed with PBS and resuspended with 100 µl binding buffer, followed by addition of 10 µl PI/FITC-Annexin V (Roche Diagnostics) and incubation at room temperature for 1 h in the dark. Flow cytometry (BD FACScan; Becton, Dickinson and Company) was used to detect apoptosis within 1 h. The cell cycle distribution and apoptosis of transfected OCI-Ly7 cells were analyzed by Cell Quest acquisition and analysis software (V6.0; BD Biosciences).