Submitted by Lauren French (download pdf)

Hek Mixed media:

 

50mls                    150mls                      250mls                    500mls

 

EMEM                                        44.5                       133.5                       222.5                       445

 

FBS(final 10%)                        5.0                           15.0                           25.0                         50.0

 

P/S/L-glut                               0.5                             1.5                            2.5                           5.0

 


-       Allow the media to warm for about one hour in the 37ºC water bath.
-       Cells frozen in DMSO can be thawed by gently shaking by hand in the water bath.
-       Gently remove cells from tube with 1mL pipette and transfer to P10.
-       Add 10 mLs of media (above)
-       Place in incubator
-       The following day, remove old media and replace with new to remove remaining DMSO

 

Notes:


-EMEM (MEM Eagle with Earle's BSS; Description: w/ L-Glutamine): Biowhittaker from Fisher Cat#:  BW12-611Q


Split Media (Hek293/Hek293-Lif/IMR32
)


-Trypsin should be diluted 4X!

Example:   For 5mls use 1.25mls Trypsin with 3.75mls HBSS

-Remove the media and add ½ amount of 4x diluted trypsin as the total amount of media removed.  If the cells are on a P10 and 10mls of media was removed add 5mls of trypsin + HBSS

-Incubate in the incubator for about 1-2 min.

-Blow off cells and place in a 15ml 0r 50ml tube

-Do not spin down

-Dilute by adding media if necessary

-Plate the cells as needed

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