SUP-B15 Cell culture protocol: based on ATCC website

Last updated: 09/28/22


            IMDM (500mL) with:

  1. 20% FBS (100mL)
  2. Pen/strep (5mL)
  3. .05mM BME (1.77uL)


Day 1:

  1. Cells are kept in the orange Liquid Nitrogen tank, Rack 3, Box 2 (counting from bottom up)
  2. Do NOT warm media in a water bath; media should be at room temp so as to not stress the cells
  3. Thaw the vial in a water bath
    1. Do NOT let the vial completely thaw in the water bathKeep lid out of water bathGently move vial side to side as thawing
    1. It should be less than 1-2min
  4. Spray tube with EtOH to decontaminate, bring into the hood
  5. Slowly add RT media to the cells
    1. For 1mL frozen cells, use 19mL of fresh media
  6. Transfer thawed cells into the tube of fresh media as they become fully thawed
  7. Transfer 20mL of media+cells into a 75cm2 Flask
  8. Label lid with the passage number, cell type, date, and initials (if new vial label with lot number)
  9. Place flask with cells in the incubator
    1. 37C, 95% Air, 5% CO2

Day 2:

  1. Check on cells by using the microscope
  2. Change media to remove any remaining cryoprotective agent
  3. Collect media + cells into a falcon tube to spin down
  4. Spin at 125xg for 5min
  5. Remove old media
  6. Add 20mL of warmed media to the falcon tube with the cells
  7. Transfer cells+media to the flask (can reuse the same flask they were in, to begin with)
  8. Return flask to the incubator
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