When thawing K562 cells, is it essential to centrifuge them or is there a way you can work with them without using the centrifuge? We are concerned about the centrifuge damaging the cells. How could you get rid of the cryo chems without centrifuge?
I haved cultured KG1a,U266 and rpmi8266cell.It is necessary to centrifuge to remove DMSO after thawing .Otherwise the cells will not live and reproduce well.
Personnally, I didn't centrifuge K562 after thawing. Just I dilute them 5-10 times in RPMI 5% FBS. The following day, you can add medium to promote cell growth.
Don't worry about breaking the cells with K562. We routinely centrifuge them down and they are really hardy...they can take pretty much anything you throw at them and we never have a problem with them. I had to centrifuge them down multiple times and then sorted them using FACs analysis, left them over night and then next day they were growing like mad.
K562s are quite hardy.
I use fresh IMDM with 10% FBS, filter & incubat in incubator at 37oC 5% CO2 to optimize pH. Thaw cells directly from cryo in 37oC water bath, swirl tube until only small bit of ice still visible, then transfer cells directly into 1/2 volume (5mL) warm media. Use remaining 1/2 dilution volume (5mL) media to rinse cryo vial. Total dilution volume 2-5 x 10e5 cells/ml. Pipet 2-3x gently to mix cells, centrifuge at 300xg - 500xg, RT, 5 min. Aspirate off media, repeat as desired. Suspend cell pellet in fresh media at 5x10e5 cells/mL, transfer to T25 flask, incubate at 37oC, 5% CO2 for minimum 24-36 hours, no longer than 48 hours.
Maintain cultures between 1 x 10e5 - 1 x 10e6 cells/mL. Double every 24 hours.
Best to use at 1/2 confluency (5 x 10e5 cell/mL), low passage #.
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