Karmella Haynes, 2013
- 70% ethanol spray bottle
- 100% FBS (50 mL aliquot), thawed at 37°C
- Complete cell culture medium with 20% FBS, if available (e.g., 20% FBS, 1% pen-strep)
- Sterile tissue culture-treated vent-capped T-25 flask, one per new culture
- One frozen vial of cells (1 mL) from the -150°C freezer
- 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 200 g for 3-5 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.
Follow-up expansion: After the cells have become 100% confluent in the T-25 flask, aspirate off the growth medium, wash the cells with 2 mL 1xPBS, trypsinize them with 0.5 mL of Trypsin buffer and transfer the entire culture into a T-75 flask with 10 mL total growth medium (10% FBS, 1% pen-strep). After the t-75 is 100% confluent, you can make new frozen stocks or passage the cells at 1:10 (or 1:5 if they are slow-growing).