Freezing Cells:

	1.	Trypsinize cells and harvest in the normal way.

	2.	Count a 200 ul aliquot and determine the total cell 
            number.  From this,  calculate the volume of media required 
            to give a final freezing density of 3.0 x 107 cells/ml.

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

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

	5.	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.

	6.	Aseptically aliquot the suspension into sterile freezing  
            vials, label each vial with the date and cell type/clone 
            number, and place the vials into a styrofoam container.

	7.	Freeze the cells overnight @ -70o C, then transfer to the 
            -135o C freezer.

Thawing Out Cells:

	1.	Remove vial of frozen cells from the -135o C freezer and 
            transfer to 37o C water bath to thaw (thawing generally 
            takes only 1-2 minutes).

	2.	Transfer the cell suspension to a sterile 15 ml tube.  
            Add appropriate media dropwise, shaking the tube well 
            after each addition.  "Top up" the tube with additional media.

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

	4.	Aspirate off the supernatant and resuspend the cell pellet in 
            12 ml of media.  Plate out the cells on a 10 cm plate 
            (use a gelled plate if plating STO's; use a 10 cm feeder plate 
            if plating ES cells).


From the Laboratory of Dr. Allan Bradley            	
Baylor College of Medicine, Houston, Texas