Haynes Lab:Notebook/Jan/2016/02/13

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Thawing Cell Lines on 2/13/16

  • Thawed one vial of U2OS cells from box A1 and one vial of U2OS 6-3 cells from box B6 according to the following protocol:

  • Pre-warm the growth medium and 100% FBS (NOT the frozen vial of cells) to 37°C in the bead bath.
  • After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood.
  • Transfer 5 mL of 100% FBS into a 15 mL conical tube (one per frozen cell vial).
  • Retrieve the frozen cell vial from the -150°C freezer.
  • Quick-thaw the frozen vial in the 37°C bead bath just until the last of the ice in the tube is thawed. Do this for no more than 2 minutes.
  • Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS. Do not mix by pipetting.
  • Spin the cells + FBS at room temperature at 1000 rpm for 3 minutes. If you have just one tube, use a counter balance of equal volume (6 mL water).
  • Get a new T-25 flask and label it with the label it with the cell line name, your initials, the date, "thawed" to indicate that this is a new culture.
  • After the cells have pelleted, bring them back to the biosafety cabinet and aspirate off the FBS, but leave about 100 uL of FBS covering the pellet.
  • Flick the tube to gently resupend the pellet in the ~100 uL FBS.
  • Stand the t-25 flask up and remove the cap.
  • Add 4 mL of growth medium to the cells. Mix by gently pipetting up and down (about 3 times).
  • Transfer the cells + medium into the T-25 flask.
  • Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion.
  • Place the flask into the 37°C incubator. The cells should adhere to the growth surface overnight.
  • Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste.