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Role of Fibroblast Growth Factors: MATERIALS AND METHODS(1)

PGC Isolation and Culture

PGCs were isolated from B6C3F1(C57Bl/6 X C3H/HeJ) mice and cultured as previously described. Briefly, single-cell suspensions were prepared by trypsinization and trituration of either caudal regions of 8.5 dpc embryos or the genital ridges of 11.5-13.5 dpc embryos. The sex of 12.5 dpc and 13.5 dpc embryos was determined by microscopic inspection for the presence of testis cords. Immu-nomagnetic PGC purification, using TG-1 antisera recognizing a PGC cell surface antigen, was performed as described previously, except that 20 ^l of TG-1 supernatant was used per preparation. ventolin 100 mcg

PGC cultures were largely performed as previously described. Briefly, cell suspensions were plated on irradiated (5000 rads) STO feeder layers in 96-well microtiter dishes that had been previously treated with 0.1% gelatin. For growth assays, approximately 0.20-0.25 equivalents of 11.5 dpc embryos were plated per well in 200 ^l of high-glucose Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS), sodium pyruvate, and glutamine. Cultures were re-fed daily. PGCs were identified by alkaline phosphatase histochemistry, as previously described. Each determination represents the average and SD of at least six wells.