Guava easycyte 12ht

At a concentration of 2.5×10⁵/mL, Raji and Raji2R cells were incubated at 37°C with either 50 µg/mL rituximab or PBS. At 1 h and 3 days after the start of incubation, the cells were transferred to 96-well plates and incubated with the RealTime-Glo MT Cell Viability Assay (Promega) following the manufacturer’s protocol. The luminescence, proportional to the number of viable cells, was measured at each time point on a Spark microplate reader (Tecan). The experiment was performed in duplicates, and the results are presented as mean ± SD.
On day 3, 2.0 × 10⁶ cells were fixed using ice-cold methanol in preparation for evaluation of apoptosis by flow cytometry analysis. A positive control was included in the study by incubating the unfixed control cells with a topoisomerase inhibitor, etoposide, for 18 h before analysis. The fixed cells were then washed and incubated with Alexa-conjugated anticleaved Poly (ADP-ribose) polymerase (PARP) antibody (BioNordika) diluted 1:100 in 5% nonfat milk for 1 h. The cells were once again washed, and the fluorescent apoptosis signal was determined by flow cytometry (Guava easyCyte12HT; Millipore).

Cells were seeded at a density of 10⁵ cells/well in 6-well plates and treated with 10 μM cisplatin and 50 μM AA. EC₅₀ was determined by the cell viability assay, under normoxia and hypoxia. Following 24, 48, and 72 h of treatment, cells were harvested by trypsinization and labeled using the Dead Cell Apoptosis Kit with Annexin-V/Alexa Fluor^® 488 and Propidium Iodide (Invitrogen/Life Technologies™, UK) as per the manufacturer’s instructions. Analysis was performed by flow cytometry on a FACSAria (BD Bioscience, UK) and Guava^® easyCyte 12HT benchtop flow cytometer (Merck Millipore, UK). A total of 10,000 gated events were analyzed and the relative fluorescence (488 nm) of Propidium Iodide plotted against Annexin-V/Alexa Fluor^® to identify healthy and apoptotic cell populations.