fcs to which 20 of culture supernatant of the b95 8 line atcc crl1612 0 1 u03bcg ml of cyclosporin a sandoz 50 u03bcg ml of gentamycin sulfate life technologies are added and distributed into round bottomed 96 well plates or 24 well plates p24 greiner they are then placed in an incubator at 37 u00b0 c 7 co 2 after 3 weeks the presence of anti rh d antibodies is sought by adcc each one of the 16 microwells of a positive p24 plate well is transferred into a new p24 well this enrichment is repeated after 10 to 15 days of culturing and each microwell is amplified in a p96 and then in a p24 the positive p96 wells are taken up and amplified in a flat bottomed p24 nunc after a few days of culturing the presence of anti rh d antibodies is sought by adcc the cells derived from one or more p24 wells are enriched in specific cells by the formation and separation of rosettes with papain treated rh d positive red blood cells one volume of red blood cells washed in 0 9 nacl is incubated for 10 minutes at 37 u00b0 c with 1 volume of papain merck solution at 1 1000th m v and then washed 3 times in 0 9 nacl the cells were then washed once in hanks solution suspended in fcs and mixed with the papain treated red blood cells in a ratio of 1 cell to 33 red blood cells the mixture is placed in a cone bottomed centrifuged tube centrifuged for 5 minutes at 80 g and incubated for one hour in melting ice the mixture is then.