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Freezing and Thawing Cultured Cells 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