CLL Cell Viability Assay with Flu Stimulation

Blood was obtained from CLL patients as defined by NCI96 criteria 28 [53 (link)] following a receipt of written informed consent under an IRB protocol approved by Saint Louis University. PBMCs were isolated from whole blood immediately following donation using Ficoll density gradient centrifugation. Isolated cells were plated in 96-well assay plates at a concentration of 10–50,000 cells (depend on patient cell numbers) per well in 100 µl of RPMI 1640 media with 10% FBS with or without 10 µM flu. The cells were cultured for 72 hrs, and then cell viability was determined using Promega’s CellTiter 96^® Non-Radioactive Cell Proliferation Assay kit (MTT) according to the manufacturer’s instructions [32 (link)]. Absorbance at 570 nm was recorded using a BioTek Epoch Reader (Winooski, VT). The rest of PBMCs from CLL patients were harvested and lysed for immunoblotting.