Recipies for Cell Fusion

Phosphate-buffered saline (PBS) (0.01 M Na2HPO4 , 0.15 M NaCl, pH 7.4):      sodium phosphate dibasic (Na2HPO4) 0.87 g      sodium phosphate monobasic (NaH2PO4 x H2O) 0.54 g      sodium chloride 8.5 g      HPLC water 1 liter      Adjust the pH to 7.4 with 1 N NaOH or 1 M HCl.

Dubelcco Modified Eagle Medium (Complete Media):      DMEM (1x liquid tissue culture medium with 4.5 g/l glucose) 70 % 500 ml      NCTC-135 medium 10% 50 ml      Fetal bovine serum 20 % 100 ml      Antibiotic/antimycotic 1% 5 ml      Fungizone 1 % 5 ml

1. Add 50 ml of NCTC-135 medium to a bottle of unopened DMEM media. Mix and transfer 50 ml to a bottle labelled “Washing Media.”

2. Add 100 ml of fetal bovine serum, 5 ml antibiotic and 5 ml fungizone to the 500 ml bottle of DMEM media. Mix, label the container “Complete Media”, and store in the refrigerator.

Washing Media:      DMEM media, unopened 500 ml       NCTC-135 media 50 ml      (Note: Washing media is usually aquired by removing media before the addition of fetal calf serum. Fetal calf serum is expensive and necessary for cell growth.)

70% ethyl alcohol:      95% ethyl alcohol 370 ml      Distilled water 130 ml

Disinfecting solution (for cleaning):      Roccall II 5 ml      HPLC water 1 liter

Disinfecting solution (for filling water pan):      Roccall II      7.5 ml      HPLC water      4 liter      HAT media      HAT supplement, 500 x      0.1 ml      DMEM Complete media      50 ml      (Note: some formulations are 50 x and should be used at the rate of 1 ml / 50 ml media)

HT media :      HT supplement, 500 x0.1 ml      DMEM Complete media      50 ml      (NOTE: some formulations are 50 x and should be used at the rate of 1 ml / 50 ml media)