cell number in hfbrx flowpaths approximately 5 liters of enriched media 5 u00d7 10 6 units il 2 and significantly fewer man hours were required to support these cultures a standard 4 hour 51 cr release assay yannelli et al 1988 canc res 48 5696 was used on days 14 and 21 to determine the functional state of the expanded lak cells the targets used were k562 daudi and a375 cells which are available from the atcc in rockville md cells from the three treatments were competent in lysing each of the target cells the composition of the cell populations was determined by flow cytometric analysis using antibodies directed to cell surface antigens the distribution of the cell surface antigens is characteristic of the different cell types the labelled antibodies used were purchased from coulter immunology hialeah fla and were specific for the antigens cd4 cd8 ta2 nkhi leull and cd25 the cells were adjusted to a concentration of 1 u00d7 10 6 ml and incubated with the antibodies at saturating dilutions for 30 minutes at 4 u00b0 c the cells were washed and analyzed on an cytofluorograph ortho diagnostics the relative properties of cytotoxic cd3 cd8 and helper cd3 cd4 t cells nk cells total and activated and total activated t cells and nk cells were similar among the three treatments over a period of 24 days the cultureware was installed according to fig3 and flushed with 5l of gibco mccoy 39 s 5a then maintained for three days at.