Remember to follow sterile technique and use 70% ethanol liberally! Warm up media and trypsin for ~20 minutes before use.
Remember: to prevent ice crystals from forming and killing your cells you want to freeze slowly and thaw quickly!
- Remove cells from liquid nitrogen storage. If not immediately thawing, place on dry ice until ready to thaw.
- Thaw the vial of cells quickly by immersing in a 37C water bath for 2-3 minutes; spray down tube liberally with 70% ethanol when removing from bath and wipe dry.
- Transfer the contents of the tube to a 15-ml conical tube containing 10 ml of growth media for the cells you are thawing; rinse the freezer vial with an additional 1-2 ml of media and add to the 15-ml conical.
- Pellet the cells at 1000-1200 rpm for 5 minutes.
- Resuspend the pellet in 10 mls of growth media by pipetting up and down until no large chunks are visible, 4-7 passes usually.
- Transfer the cells to a 10 cm dish labeled with the passage number (while historical passasage may not be known, keep track of your passage number for reference). Thawed cells are labeled “P0”. Check cell viablity under the microscope, healthy cells will be bright and refractile. Grow cells at 37C with the recommended CO2 concentration (5% for our cells).