Facsdiva software version 6

For flow cytometry analyses and cell sorting tumor spheres were dissociated as single cells, washed and incubated with the appropriate dilution of control or specific antibody. Antibodies used were V450 Mouse Anti-Human CD44 (561292), PE-Cy7 Mouse Anti-Human CD45 (557748), PE Mouse Anti- Human CD146 (550315), PE-Mouse Anti-Human CD24 (555428), FITC Mouse Anti-Human CD90 (561969), FITC-Mouse Anti-Human EpCAM (347197), APC-Mouse Anti-Human CD10 (332777), (all from BD Bioscience Bedford, Franklin Lakes, NJ) and Mouse monoclonal antibody [5D3] to Cytokeratin 8 + 18 (Abcam Cambridge,UK, 17139). After 45 min incubation, cells were washed. Analysis was performed using a FACS Canto flow cytometer (BD Biosciences). Cell Sorting was performed by FACS ARIA cytometer equipped with three lasers (488, 633, 407 nm) (BD Biosciences). All the cytofluorimetric acquisitions were analyzed by BD FACSDiva Software version 6.1.3 (BD Biosciences). For the sorting, cells were incubated with antibodies for 1 h then washed in PBS. Antibodies were used following manufacturing protocol indication for sorting experiment. TO-PRO3 (Thermo Fisher) dye was used for viability evaluation and used following manufacturing protocol indication. All the cytofluorimetric acquisitions were analyzed by BD FACSDiva Software version 6.1.3 (BD Biosciences).

Flow cytometry was used to determine various types of cell death including early and late stage apoptosis as well as necrosis. Annexin V-FITC and propidium iodide (Annexin V-FITC apoptosis detection kit, eBiosciences, Inc., San Diego, CA, USA) [33 (link)]. Cells were seeded in 24-well plates at a density of 5 × 10⁵ cells/ml per well and incubated for 24 h. Cells were treated with the crude extracts at a concentration of 1 × IC₅₀ and 2 × IC₅₀, obtained from the antiproliferation study at 12 and 24 h. Afterward, cells were harvested by centrifugation (Wisds’ Laboratory Instruments, Korea) at 1677g for 5 min then the supernatant was removed. The cells were washed with 200 µL of 1x binding buffer. After removal of the supernatant, 95 µL of binding buffer and 5 µL Annexin V-FITC were added and the mixture incubated in the dark for 15 min at room temperature. Then 95 µL of binding buffer and 5 μL of propidium iodide (final concentration of 2 μg/mL per cell sample) were pipetted into an Eppendrof tube and the mixture incubated for 15 min in the dark at the room temperature. Cells were re-suspended in 200 μL of binding buffer. The stained cells were analyzed immediately by flow cytometry (BD FACSCanto II, Franklin Lakes, NJ, USA) by FACSDiva software version 6.1.3 (BD Biosciences, San Jose, CA, USA).

Neurons and glial cells of immortalized gba^−/− and gba^+/+ neuronal cultures were separated by FACS. Cells of each genotype were labeled with FITC hamster anti-rat CD29 and PE rat anti-mouse CD24 (BD Biosciences, San Jose, CA, USA). Single-stained and unstained cells were used as a control. Cells were sorted using a BD FACSAriaII cytometer (BD Biosciences). Results were analyzed with FACSDiva software version 6.1.3 (BD Biosciences).