Isolation of T4 cells from xenograft tumors

1 Put mouse down 2 Wash skin by dipping mice into ETOH 3 Remove tumor and put into 5ml PBS containing Fungizone and Penstrep 4 Swirl around to rinse tumor and pour on to a sterile plate 5 Remove tumor from PBS place on sterile plate or lid 6 Remove any skin-rinse a second time if needed 7 Chop into fine pieces with scalpel blades 8 Transfer using blade into 2ml H14 media containing Fungizone and Penstrep 9 Spin down 800rpm 10 Remove media leaving ~300ul 11 Add 2ml .25% trypsin and place on rocker for 5min 12 Spin 800rpm for 5min 13 Resuspend in DMEM/F12 with10% FCS 14 Spin 800rpm for 5min 15 Resuspend in H14 media containing Fungizone and Penstrep 16 Plate onto vitrogen coated flask of plate (volume of media and size of flask depend on tumor size I used a 25cm2 flask)

17 change media the next day and keep close eye out for contamination