Lsrii sorp system

U-2 OS cells were seeded at 3000 cells/well into 96-well plates (flat bottom) and treated as indicated for 48 h. After washing in annexin V-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4), cells were incubated for 15 min at RT in 50 µL annexin V-binding buffer containing propidium iodide (BioLegend, San Diego, CA, USA) and Alexa Fluor 488-conjugated annexin V (Thermo Fisher Scientific, Waltham, MA, USA). For the image-based analysis of U-2 OS cells, the adherent cell-by-cell module (IncuCyte S3) was used, allowing assessment of green and red fluorescence per each individual cell. For flow cytometric analysis of suspension B-cells, WEHI-231 and A20 were seeded at 100.000 cells/well into 96-well plates (round bottom) and treated as indicated for 24 h. Cells were washed once with Annexin V-binding buffer and incubated at RT in 50 µL Annexin V-binding buffer containing Alexa Fluor 488-conjugated annexin V and Live/Dead Fixable Blue Dead Cell stain (Thermo Fisher Scientific, Waltham, MA, USA). Flow cytometric measurements were performed using the BD LSRII SORP system (BD Biosciences, Haryana, India) and data were analyzed with FlowJo software (BD Biosciences, Haryana, India).

For cell cycle analysis, U-2 OS cells were treated for 24 h, as indicated. Cells were harvested by centrifugation and pellets washed twice with ice-cold PBS. Cells were fixed with 70% of ice-cold ethanol for 30 min followed by washing with PBS twice. 50 µg/mL RNase A and 50 µg/mL propidium iodide solution was added to the remaining cell suspensions. Subsequently, cell fluorescence was measured using the BD LSRII SORP system (BD Biosciences, Haryana, India). Fluorescence intensity analyses were performed with FlowJo software (BD Biosciences, Haryana, India).