Stable EGFR and Erbb2 Conditional Knockdown Mouse Skin Keratinocyte (B1B2F3) Cell Line

Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Thaw the cells with PriGrow XI (TM011) + 8% FBS + 60 µM CaCl₂ (added fresh each time) + 1% Penicillin/Streptomycin Solution (G255)

37.0°C

8% CO₂ for optimal attachment. After 24h

change media to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255)

37.0°C

8% CO₂ Steps to thaw the cells: 1. Thaw the cells in RT water until only a few ice crystals are left 2. Put the cells in 5 ml of RT PriGrow XI (TM011) + 8% FBS 3. Centrifuge the cells 1500 rpm for 5 min (need this step to remove inhibitors for attachment) 4. Resuspend the pellet in RT Prigrow XI (TM011) + 8% FBS + 1% P/S with freshly added 60 µM CaCl₂ and put the cells in a culture vessel at 37.0°C

8% CO₂ 5. After 18h – 24h

change to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255)

37.0°C

8% CO₂ for growth of the cells.