REEVALUATION OF THE CULTURE CONDITION OF POLYMORPHONUCLEAR CELLS FOR THE STUDY OF APOPTOSIS INDUCTION

The culture conditions of human peripheral blood polymorphonuclear leu kocytes (PMN) in the study of apoptosis induction were re-evaluated. T he changes in the relative viable cell number of PMNs after tumor necr osis factor (TNF) treatment were colorimetrically investigated using a cell counting kit. The relative potency of PMNs to produce the supero xide anion (O-2(-)) was measured as the reduction of color intensity b y addition of superoxide dismutase (SOD). When the PMNs were cultured in conventional RPMI1640 medium supplemented with 10% fetal bovine ser um (FBS), the stimulation effect of TNF on O-2(-) generation by PMNs w as observed only for the first 6 hours. When FBS was replaced with hum an serum, the effect of TNF was maintained for longer incubation perio ds. Prolonged incubation of PMNs spontaneously produced large DNA frag ments, and the extent of DNA fragmentation was relatively smaller in h uman serum-containing medium. TNF, LPS, hyperthermia or potassium thio cyanate slightly accelerated the production of large DNA fragments, as well as the induction of trace amounts of internucleosomal DNA cleava ge in PMNs, which became detectable only after concentration by fracti onal isopropanol precipitation. The present study suggests the importa nce of the use of human serum rather than conventional FBS for the stu dy of apoptosis induction in PMNs.