Re-evaluation of the culture condition of polymorphonuclear cells for the study of apoptosis induction.
The culture conditions of human peripheral blood polymorphonuclear leukocytes (PMN) in the study of apoptosis induction were re-evaluated. The changes in the relative viable cell number of PMNs after tumor necrosis factor (TNF) treatment were colorimetrically investigated using a cell counting kit. The relative potency of PMNs to produce the superoxide anion (O2-) was measured as the reduction of color intensity by addition of superoxide dismutase (SOD). When the PMNs were cultured in conventional RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), the stimulation effect of TNF on O2- generation by PMNs was observed only for the first 6 hours. When FBS was replaced with human serum, the effect of TNF was maintained for longer incubation periods. Prolonged incubation of PMNs spontaneously produced large DNA fragments, and the extent of DNA fragmentation was relatively smaller in human serum-containing medium. TNF, LPS, hyperthermia or potassium thiocyanate slightly accelerated the production of large DNA fragments, as well as the induction of trace amounts of internucleosomal DNA cleavage in PMNs, which became detectable only after concentration by fractional isopropanol precipitation. The present study suggests the importance of the use of human serum rather than conventional FBS for the study of apoptosis induction in PMNs.
Hiroi
M
M
Department of Dental Pharmacology, Meikai University School of Dentistry, Saitama, Japan.
Tajima
M
M
Shimojima
T
T
Kashimata
M
M
Miyata
T
T
Sakagami
H
H
D000818:Animals; D001769:Blood; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D016895:Culture Media, Serum-Free; D053938:DNA Fragmentation; D004926:Escherichia coli; D018922:HL-60 Cells; D006358:Hot Temperature; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D016966:Porphyromonas gingivalis; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000097:blood*; D013861:Thiocyanates / Q000633:toxicity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D003470:Culture Media; D016895:Culture Media, Serum-Free; D008070:Lipopolysaccharides; D013861:Thiocyanates; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D013482:Superoxide Dismutase; C009941:potassium thiocyanate