Annexin 5 detection kit

Apoptotic cells were visualized using Annexin V Detection Kit according to the manufacturer's instructions (BD Pharmingen). Briefly, cells were treated with dinaciclib 50nM for 24 hours followed by incubation with fluorescein isothiocyante (FITC)-conjugated Annexin V and propidium iodide (PI) at room temperature for 15 minutes. After 15-minute incubation, samples were analyzed using AccuriC6 flow cytometer (BD Accuri Cytometers) and cell quest software. Cell-cycle phase distribution was determined in 70% alcohol-fixed cells stained with 5ul PI-RNase A staining buffer (Invitrogen) for 30 minutes at room temperature, followed by analysis using AccuriC6 flow cytometer (BD Accuri Cytometers) and cell quest software.

Spleen B cells were isolated (B cell isolation kit, Miltenyi) and cultured overnight in the presence of 25 μg/ml LPS (Escherichia coli 055:B5, Sigma-Aldrich, Diegem, Belgium) to maintain sufficient cell survival and support antigen presentation. Dead cells were removed by Ficoll centrifugation (Lympholyte-M, Cedarlane Labs, Atlanta, GA, USA) and remaining B cells were stained with Cyto-ID Red long-term cell tracer kit (Enzo Life Sciences, Lausen, Switzerland) following manufacturers’ instructions. B cells were then cocultured for 18 h with CD4⁺ T cells (ratio B:T, 1:5) in the presence of indicated peptide (2 μM, added to the culture media). Annexin V APC was used to detect cell death in B cells (Annexin V detection kit, BD Biosciences) according to manufacturers’ instructions. Gated B cells were then analyzed for Annexin V binding on flow cytometer. For inhibition of granzyme-B (GZB) activity, Z-AAD-CMK (Calbiochem/Merck, Overijse, Belgium) was added at 20 μg/ml during the entire coculture period. Inhibition of FasL was performed with functional grade anti-mouse CD178 antibody (clone MFL3, eBioscience) at 20 μg/ml during the coculture period.

Apoptosis was assessed with the Annexin V detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. Cells were analyzed by flow cytometry on a FACS Verse instrument (BD Biosciences).

Cells were stained with Annexin V detection kit (BD Biosciences) according to the manufacturer’s instructions, coupled with propidium iodide (PI; Sigma) staining and analyzed by flow cytometry.

DAOY cells were seeded in 6-well plates at a density of 200,000 cells per well 24 h prior to transfection, and maintained in 10% DMEM supplemented with 1% penicillin-streptomycin, and 1% L-glutamine for 24 h before transfection. Cells were transfected in serum-free DMEM, with 1% penicillin-streptomycin and 1% L-glutamine. At 24 h after transfection, cells were irradiated with a one-time radiation fraction of 10 Gy. Cells were incubated at 37°C for 24, 48 h and 72 h after irradiation. At the end of each incubation period, cells were collected for analysis of cell cycle distribution by flow cytometry. MTT analysis was done to determine the effect on cell growth 96 h post-transfection. Furthermore, the percentage of cells undergoing apoptosis was analyzed following EphB1 knockdown and ionizing radiation exposure 72 h post-transfection using an Annexin V detection kit (BD Pharmingen), according to the manufacturer's instructions. Analysis was done by Becton Dickinson FACS Calibur system.

Cells were seeded at a density of 5 × 10³ cells/well in a 96-well plate, and the role of Prx II and miR-122 in A549/GR CSC proliferation was studied using the EZ-Cytox Kit (DoGenBio, Korea. Catalog number: EZ-3000), according to the manufacturer’s instructions. The absorbance in each well was measured at 450 nm. To detect apoptosis, cells were prepared using the Propidium Iodide (PI), Annexin V Detection Kit (BD Biosciences), per the manufacturer’s instructions, and analyzed by flow cytometry (FACSCalibur, BD Biosciences).