Flow cytometric evaluation of apoptosis, necrosis and recovery when culturing monocytes

After developing and applying a method for cryopreserving monocytes, we fou nd a substantial cell loss when culturing these cells. Monocytes were isola ted from blood donors by density gradient centrifugation, purified by elutr iation and cryopreserved. Thawed cells were cultured in ultra low attachmen t wells and studied with Annexin V, Propidiumiodide, Dihexyloxacarbocyanine (DiOC(6)(3)), bromolated deoxyuridine triphosphate nucleotides (Br-dUTP), DNA ploidy and DNA ladder methodologies. The main cell loss was within the first 24 h and recovery on day 7 was 35-40%. The first 2-6 h of culture wer e found to be crucial for determining which cells survive. Initially (2-4 h ), apoptosis was the main feature but after 6 h, necrosis dominated. Two po pulations of cells developed after 24 h: "A" consisting of larger cells wit h low levels of apoptosis and necrosis signals and population "B" comprisin g smaller cells with a high expression of necrotic but low levels of apopto tic signals. Signs of DNA fragmentation were slight. These early, dynamic c hanges may be important for the interpretation of experimental results when investigating monocytes in culture. (C) 2001 Elsevier Science B.V. All rig hts reserved.