The procedure for trophoblast isolation was based on the method of Kliman et al. with modifications. Briefly, the basal plate was removed, and approximately 50 g of villous tissue was harvested and teased into small fragments. Villous trophoblast cells were released by four sequential trypsin digestions (trypsin grade III; Sigma Chemical Co., St. Louis, MO). DNase I (grade II; Boehringer Mannheim, Indianapolis, IN) was used to prevent cellular aggregation. The first digestion was routinely discarded, as it contained few trophoblast cells and mainly red blood cells and leukocytes. The digests were layered over FBS and centrifuged to remove cellular debris and inactivate trypsin. antibiotic levaquin
The trophoblast cells were first separated from other cell types by Percoll (Pharmacia and Upjohn, Kalamazoo, MI) gradient centrifugation and then further purified using magnetic beads (PerSeptive Diagnostics, Cambridge, MA) coupled to anti-human leukocyte antigen-Class 1 (HLA-A,B,C) (Dako, Carpinteria, CA) for third-trimester placentas, and BioMag anti-CD45 antibody (PerSeptives Diagnostics) for first-trimester placentas. Purity was routinely > 97% as determined by immunocytochemical criteria including positive staining for cytokeratin, and viability was routinely > 95% by Trypan Blue staining. Freshly isolated cells were either snap frozen in liquid nitrogen and stored at – 80°C until extraction of RNA or protein, or were immediately placed in 75-cm2 tissue culture flasks (8-10 X 106 cells per flask) for a 30-min preincubation at 37°C in RPMI-1640 medium containing 0.5% BSA for analysis of EPO-R phosphorylation.