Revision as of 00:13, 15 May 2012 by Christopher C Vanlang (New page: : Hey Kedar, : : No problem. For the freezing cell protocol it is pretty simple. First make : sure the cells are crowded but very healthy before freezing. Make a : 10%DMSO solution from ...)
- Hey Kedar,
- No problem. For the freezing cell protocol it is pretty simple. First make
- sure the cells are crowded but very healthy before freezing. Make a
- 10%DMSO solution from your regular RPMI 10% FBS, Glu, P/S, b-me media and
- chill it on ice. Also prechill cryotube (for fast chilling I stick them in
- the -80oC). For a crowded 25mL dish I will prepare 5 vials. Spin down the
- cells at 1000-1200 rpm for 5'. Dump media. Resus the pellet very well (no
- clumps) in 5mL of DMSO media and put 1mL/vial.Immediately put into a box
- full of cotton that was at room temperature. (The idea is to have the
- temperature drop slowly) Then put into -80oC freezer. Next day transfer on
- dry ice to liquid nitrogen for long term storage. A week later thaw one
- vial to see if the freezing was done well and the cells are viable.
- For thawing, aliquote 10mL of RPMI 10%FBS in a falcon tube, prechill on
- ice. Close centrifuge lid to keep at 4oC. Warm the cryotube in hand until
- there is only a small chunk of ice left, pipet some RPMI 10%FBS media into
- the cryotube which should melt the little ice chunk and transfer all into
- the 10mL tube. Spin (same speed, time) immediately, remove media. Wash one
- more time with 10mL media. Plate on a 12 well plate or 5mL dish to keep
- cell density relatively high. Supplement with an additional 10% of FPBS.
- Wait for 3 hours. Check under microscope again, if you think the cells are
- too crowded and they will not have enough food thru the night, should move
- to a bigger dish.