suggested protocol membranes were washed once at 65 u00b0 c for 30 minutes in 2 u00d7 ssc containing 0 1 sds and then at 65 u00b0 c for 30 minutes in 0 2 u00d7 ssc containing 0 1 sds in the elisa cell culture supernatant 10 5 cells equivalent ml from each cell type was recovered 48 hours after medium exchange and assayed for il 5 ii13 and gm csf production using quantikine kits r amp d systems minneapolis minn usa specific for each cytokine according to the manufacturer 39 s suggested protocol results indicated that notch2 urokinase and nf il 3 were up regulated in lcl or nhl cell lines as well as in the hd derived cell lines but overexpression of il 13 il 5 and gm csf was restricted to the hd derived cell lines il 5 and gm csf expression and secretion could be demonstrated only in l428 and kmh2 cells but mrna expression and secretion of il 13 could be detected in all three hd derived cell lines hdlm2 cells secreted a moderate amount of il 13 27 pg ml while l428 and kmh2 cells secreted significantly higher levels of 4800 pg ml and 6100 pg ml respectively to confirm that il 13 was expressed by the rare h rs tumor cells in situ hybridization was carried out with sense and antisense rna probes for il 13 using lymph node biopsies from four untreated patients newly diagnosed with classical nodular sclerosis hd a benign.