Swartz:Protocols/A20 cells

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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.