PGC cell suspensions were obtained as described above, but trypsinization was terminated after 5 min at 37°C by addition of one drop of FBS. The cell suspensions were quickly washed twice in 1 ml binding buffer (a 1:1 mixture of DMEM and Ham’s F-12 supplemented with insulin and transferrin [Sigma Chemical Co., St. Louis, MO] and 0.1% BSA). Cell suspensions were diluted to 6 X 106 total cells/ ml. Cell suspension (100 ^l) was incubated with 750 000 cpm of 125I-bFGF (800-1200 Ci/mmol; Amersham, Arlington Heights, IL) with agitation for 45 min at 4°C. buy birth control online
Where indicated, some samples had been previously supplemented with 0.5 ^g unlabeled recombinant human bFGF (Life Technologies, Bethesda, MD). Suspensions were pelleted through 200 ^l of cold FBS, resuspended in 0.2 ml of a 1: 1 mixture of binding buffer and FBS, and pelleted in a cytospin for 10 min at 700 rpm onto 3-aminopropylethoxy-silane-treated microscope slides (Digene Corp., Silver Springs, MD). The slides were fixed for 10 min in cold 70% ethanol, followed by 10 min in 4% paraformaldehyde in PBS. PGCs were histochemically stained for alkaline phosphatase as previously described.