Freezing cells

2/15/06

1. Use 10cm plates that are essentially confluent, but not overly crowded. For 293T cells, split 2d earlier at 1:10.

2. Make up freezing medium (you will need 1mL/plate). This should have base medium normally used for the cells being frozen (ex: DMEM), normal concentration of serum (ex: 10%), and glutamine. For some sensitive cell lines (ex: PC12) you may choose to leave out the pen/strep, however I generally leave it in. Then add 10% DMSO (so add 1mL DMSO to 9mL medium). Pre-chill the freezing medium on ice for at least 20’ before adding cells to freeze.

3. Prewarm normal growth medium, 1X Ca/Mg-free HBSS, and trypsin.

4. Remove medium from cells to freeze, wash 2X with HBSS, and add 2mL trypsin. Return to incubator for about 5’.

5. Check in scope to make sure cells are coming up off the dish, then add 10mL regular cell growth medium, pipette cells up off plate and place in 15mL conical.

6. Spin down 5’ at 2K.

7. Remove medium from cells, and resuspend cell pellet in 1mL freezing medium.

8. Immediately move cells to a prelabeled freezing vial (record cell line, passage, date, and your initials). 1 dish/tube.

9. Once all cells are in freezing vials, place inside a styrofoam 15mL tube rack and put in –80 overnight labeled with the date and your initials. Write a note to move to liquid N2 freezer next day.

10. Next day move cells to liquid N2 freezer and document their location in the N2 log book.