Thawing Frozen Vials
- Quickly warm vial to 37°C in pre-warmed water in incubator.
- Immerse in ethanol to sterilize.
- Transfer contents of vial to 15 mL Falcon tube, add 5 mL medium.
- Centrifuge at 1000 g, 2 minutes to pellet cells.
- Aspirate medium, add 5 mL fresh medium.
- Grow cells at 37°C in 5% CO2 incubator.
- Examine flasks under inverted microscope for morphology.
- Feed Monday, Wednesday, Friday by removing medium and replacing with an equal (or slightly larger) volume of fresh medium, pre-warmed to RT.
- Subculture when at near-confluence, at least once per week.
- Remove all media.
- Rinse with 5 mL/dish of PBS to remove all traces of serum.
- Add 1 mL HBSS-Trypsin-EDTA, stand for several seconds, and remove.
- Incubate at RT for 5-15 minutes, until cells appear detached and clumping under microscope, then tap gently with palm of hand to dislodge cells.
- Add 5 mL fresh medium, washing cells off flask wall, and collect in a 50 mL Falcon tube, reserving two drops in an Eppendorf tube for counting.
- Aspirate into new flasks (3×106 cells/75 cm2 flask).
- Top up medium in new flasks to an appropriate level (12 mL).
Counting and Viability Check
- Remove 40 µL of culture from prepared Eppendorf tube, transfer to another Eppendorf tube containing 40 µL 0.4% Trypan blue (Gibco 15250-061).
- Mix, then slowly load into hemocytometer.
- Using 40× objective, count all non-blue cells in 5×5 central square.
- Multiply count by 0.02 to find cell density, and again by volume for cell count.
- Count all blue cells in square.
- Divide non-blue count by (non-blue + blue) count to find viability.
- Clean hemocytometer.
- Follow above procedure for trypsinization.
- Centrifuge resulting suspension.
- Aspirate supernatant, resuspend in 1 mL freezing medium per million cells.
- Aliquot into cryogenic tubes.
- Place in Nalgene freezing vessel (containing isopropanol) at RT.
- Transfer to –70°C freezer overnight.
- Transfer to liquid N2.
Harvesting for Protein
- This protocol was modified from Santa Cruz protocol.
- Remove all media.
- Rinse flask with 5 mL PBS at RT.
- Add 600 µL ice cold RIPA (or another lysis buffer, including inhibitors) per flask.
- Collect with a cell scraper and syringe with 21G needle; transfer lysate to Eppendorf tube on ice.
- Wash flask with 300 µL ice cold RIPA, collect with needle, and add to Eppendorf.
- Shear DNA by drawing six times through needle.
- Continue with protein preparation protocol.