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Protocols: ES Cell Culture - Gene Targeting - Protocols


	All procedures should be carried out using sterile techniques at 
all times.  Media for STOs/ SNL 76/7 cells is DMEM (high glucose, no 
pyruvate), 7% FBS and 1X GPS.

1.	Gelatinize plates, at room temperature for 2 hours.

2.	Aspirate media off the 15-cm plates.

3.	Wash cells 1X with PBS.  Aspirate off the PBS.

4.	Add 2.5 ml of Trypsin to each plate (15 cm), cover the surface entirely.

5.	Incubate plates @ 37 C, for 5 min.

6.	Add 5 - 6 ml of feeder media (DMEM, 7% FBS, 1XGPS) to each 
plate.  This inactivates the trypsin.

7.	Harvest cells by pipeting up and down the cell suspension and 
transfer to sterile 50 ml centrifuge tube.  Repeat the process and pool 
all plates into 1 tube.  You can pool 5 x 15 cm dishes into 1 x 50 ml 
centrifuge tube.

8. Count before spin, with the Coulter Counter. 

9.	Next, dispense exactly 6- 7 mls of cell suspension per each 15 ml 
tubes;  you will have as a result 7 x 15 ml tubes per every 50 ml 
centrifuge tube. 

10.	Cells are now ready to be gamma irradiated.  You need to 
administer 6,000 rads.  If you have not use the Irradiator, you need to 
SOMEONE SHOW YOU HOW.  Irradiate cells with the Irradiator:  Gammacell 
1,000 @ The Immunology Dept., Room M-920, DeBakey building, 9th floor 
(Attention: Chris Arhelger, if you have any problems with the Irradiator, 
contact Ms. Arhelger.)
o		Before you irradiate cells, always check that the 
Turn-table is ON; that the dose factor and the time (in minutes) are correct.
o		Dose Factor = 100.0 * (October, 1998)			
Time:  6 Minutes

Each minute = 1,000 rads @ the dose factor of 100.  This will be equal to 
a total of 6,000 rads. 

11.	After cells are irradiated,   RETURNED all the 7x 15 ml 
centrifuge tubes to 1 x 50 ml tube.  Do the same for the second set of 
tubes.   Now determine the total cell number.   

12.	Determine the total cell number.  Calculate the volume of media 
required to give a final freezing density of 4.2 x 107 cells/ml (reg. 
Feeders).   Each vial = 1 ml = 4.2 x 107 = 10 x 10 cm feeders.

	13.	Collect the cells by centrifugation @ 1,000 rpm for 7 minutes.

	14.	Aspirate off the supernatant and resuspend the pellet in 
1/2 the volume calculated. Use media appropriate for the cells being 
frozen (i.e., M15 for ES cells or 7% FCS, 1% GPS for STO's).   ADD the 
STO�s media FIRST.

	15.	Dilute the cell suspension 1:1 with 2X Freezing Media 
(60% DMEM, 20% FCS, 20% DMSO; freshly prepared).  Add the media dropwise, 
mixing well after each addition.

	16.	Aseptically aliquot the suspension into sterile freezing 
vials, label each vial with the following:  IRRAD, Date and cell 
type/clone number, Passage number and place the vials into a 
cryo-freezing container or styrofoam container.

	17.	Freeze the cells overnight @ -70o C, then transfer to the 
-135o C freezer or Liquid Nitrogen Freezer.

*  The Dose Factor is adequate for only one year, so annually the 
Irradiator needs to be calibrated.  (For 1996, after calibration, the 
dose factor = 78.0.   For 1995, dose factor= 71.4.   For 1994, the dose 
factor was= 66.0.  FOR OCTOBER 1998 = 100.