Subculturing Human Umbilical Vein Endothelial Cells (HUVEC)
REAGENTS:
1. Hank's Balanced Salt Solution without Ca++ and Mg++ (HBSS), from Biowhittaker, catalogue #10-543.
2. Trypsin-Versene Mixture (0.5% trypsin - 0.02% EDTA), from Gibco, Cat. #610-5300 or Biowhittaker, Cat. #17-161. Dilute 1:1 with HBSS before use.
3. Nutrient Medium, consisting of 20% Fetal Calf Serum from Gibco, Cat. #200-6140, 80% Medium 199 (M199) buffered with 25mM HEPES from Biowhittaker, Cat. #12-118, and supplemented with fresh 2mM L-Glutamine (final concentration) from Biowhittaker, Cat. #17-605E, and with 100 U/ml K-Penicillin G with 100 mcg/ml Streptomycin Sulfate from Biowhittaker, Cat. #17-719R or Gibco, Cat. #600-5140. On the day of use, add 50 mcg/ml Endothelial Cell Growth Supplement (ECGS) from Biomedical Technologies, Inc., Cat. #BT-203, (See Protocol #2), and 100 mcg/ml Heparin from Sigma Chem. Co., Cat. #H-3933 (See Protocol #3).
4. 0.1% Gelatin, from Difco, Cat. #0143-02, dissolved in pyrogen-free, tissue culture grade water and autoclaved.