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Faculty Research Labs

Routine Culture of Adherent Cells

Thawing Frozen Vials

  1. Quickly warm vial to 37°C in pre-warmed water in incubator.
  2. Immerse in ethanol to sterilize.
  3. Transfer contents of vial to 15 mL Falcon tube, add 5 mL medium.
  4. Centrifuge at 1000 g, 2 minutes to pellet cells.
  5. Aspirate medium, add 5 mL fresh medium.
  6. Plate.

Routine Culturing

  1. Grow cells at 37°C in 5% CO2 incubator.
  2. Examine flasks under inverted microscope for morphology.
  3. Feed Monday, Wednesday, Friday by removing medium and replacing with an equal (or slightly larger) volume of fresh medium, pre-warmed to RT.
  4. Subculture when at near-confluence, at least once per week.

Subculturing

  1. Remove all media.
  2. Rinse with 5 mL/dish of PBS to remove all traces of serum.
  3. Add 1 mL HBSS-Trypsin-EDTA, stand for several seconds, and remove.
  4. 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.
  5. 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.
  6. Aspirate into new flasks (3×106 cells/75 cm2 flask).
  7. Top up medium in new flasks to an appropriate level (12 mL).

Counting and Viability Check

  1. Remove 40 µL of culture from prepared Eppendorf tube, transfer to another Eppendorf tube containing 40 µL 0.4% Trypan blue (Gibco 15250-061).
  2. Mix, then slowly load into hemocytometer.
  3. Using 40× objective, count all non-blue cells in 5×5 central square.
  4. Multiply count by 0.02 to find cell density, and again by volume for cell count.
  5. Count all blue cells in square.
  6. Divide non-blue count by (non-blue + blue) count to find viability.
  7. Clean hemocytometer.

Freezing

  1. Follow above procedure for trypsinization.
  2. Centrifuge resulting suspension.
  3. Aspirate supernatant, resuspend in 1 mL freezing medium per million cells.
  4. Aliquot into cryogenic tubes.
  5. Place in Nalgene freezing vessel (containing isopropanol) at RT.
  6. Transfer to –70°C freezer overnight.
  7. Transfer to liquid N2.

Harvesting for Protein

  1. This protocol was modified from Santa Cruz protocol.
  2. Remove all media.
  3. Rinse flask with 5 mL PBS at RT.
  4. Add 600 µL ice cold RIPA (or another lysis buffer, including inhibitors) per flask.
  5. Collect with a cell scraper and syringe with 21G needle; transfer lysate to Eppendorf tube on ice.
  6. Wash flask with 300 µL ice cold RIPA, collect with needle, and add to Eppendorf.
  7. Shear DNA by drawing six times through needle.
  8. Continue with protein preparation protocol.