20.109-Protocols/Thawing J1

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Similar to that described on atcc.org for product SCRC-1010, with some changes. Briefly,

  1. Coat the culture flask to be used with pre-warmed gelatin for at least 10 min.
    • Use 5 mL for a T75, or 1 mL for one well of a 6-well plate.
  2. Frozen J1 are stored in the Niles lab liquid nitrogen vessel. Bring an aliquot to lab, and thaw it in the 37 °C water bath.
    • Gently shake the vial, while submerging only the body - keep the cap above the surface of the water to prevent contamination.
    • Limit the immersion time to the minimum needed (~2 min).
  3. Pipet the 1 mL of cells into a conical tube containing 8 mL of pre-warmed culture medium. Let the cells fall drop by drop.
  4. To wash any remaining cells out of the vial, use another 1 mL of medium.
  5. Spin the cells down for 5 min at 500 g.
    • Our centrifuge does not work well at lower rcf.
  6. Resuspend the cells in an appropriate volume.
    • For a T75, culture in 15 mL total. For one well of a 6-well, use 6 mL.
  7. If you use a T75...
    • Change the medium every day.
    • A 5M aliquot will probably need about 3-4 d of growth before splitting.
  8. If you use a 6-well...
    • Change the medium after the first day.
    • A 5M aliquot will probably need about 1-2 d of growth before splitting to a single T75.
    • After 1-3 days, the cells should be propagating normally and ready for a typical split.
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