The final digestate was passed through a 23G needle to dissociate the remaining cell clusters. The isolated cells were centrifuged at 1500 rpm for 5 min at 4°C, and washed in Ham F-10 medium. This procedure was repeated three times and the final cell pellet was resuspended (1 g of tissue/ml) in 20% Percoll (Sigma), layered on a 60%:50%:40% discontinuous Percoll gradient, and then centrifuged at 22000 rpm for 20 min at 4°C. The top cell layer was collected, washed, resuspended in Ham F-10, and centrifuged at 1500 rpm for 5 min at 4°C.
After repeating this procedure, the resulting cell pellet was resuspended in 40% Percoll (1 g of tissue/ml), layered on a 55%:50%:40% discontinuous Percoll gradient, and centrifuged at 22 000 rpm for 20 min at 4°C. The top cell layer was washed twice in serum-free Ham F-10, and then centrifuged at 500 rpm for 5 min at 4°C. The cell pellet was resuspended in Ham F-10 plus 10% SCS and the decidual cells were counted in a hemocytometer. Trypan blue exclusion identified more than 95% of isolated cells as being viable.
Isolated decidual cells (5 X 105 cells/ml) were suspended in Basal Medium (BM), which is a phenol red-free 1:1 (v/v) mix of DMEM (Invitrogen, Carlsbad, CA) and Ham F-12 (Flow Laboratories) with 100 U/ml penicillin, 100 ig/ml streptomycin, and 0.25 ig/ml fungizone, and supplemented with 10% SCS.