Method: Preparation of Lymphocyte Cell Pellet for Storage

June 10, 1990

Rosalie Veile

Purpose:

• Following propagation to 1 X 108 cells, lymphoblastoid cells are conveniently stored at -80 degrees C to preserve the high molecular weight DNA in the cells until the DNA is purified. This procedure describes the steps required to harvest and freeze the cells for long term storage.

Time required:

• 2-3 hours to prepare 12-15 cultures for storage.

Procedure:

1. Aspirate the growth media from the lymphoblastoid cell culture to the 40 ml mark on the T-75cm2 flask.

   
   

2. Resuspend the cells in the flask by shaking gently. Remove 200 祃 of the cell suspension and determine the cell count (refer to cell counting procedure). Transfer the cell suspension either by decanting or pipetting to a 50 ml conical centrifuge tube.

   
   

3. Centrifuge the tubes containing cells for 10 minutes, 1200 rpm, at room temperature using the T-J6 centrifuge. Do not apply the brake at the end of the centrifuge run.

   
   

4. Aspirate the supernatant above the cell pellet. Resuspend the cells with 10 ml of PBS.

   
   

5. Label a 15 ml tube with the date, kindred#, cell line #, and cell count. Transfer the cell suspension to the labeled 15 ml centrifuge tube, centrifuge again for 10 minutes and aspirate the supernatant.

   
   

6. Transfer the tube to a -80 degrees C Revco freezer and record the rack location on the cell line growth record sheet.

Reference:

Dr. R. Todd, Department of Psychiatry