Method:ReactivatingCellLinesandCellGrowthforDNAPreparation

Purpose:

• Cell lines are reactivated and grown to a count of 1 x 108 cells. The cells are pelleted and stored frozen at -80 degrees C prior to DNA extraction.

Time required:

• 15-20 minutes to begin growing 2-4 cryovials

Procedure:

1. Frozen cells should be thawed quickly. Remove the cryovial from its long term storage container in the -135 degrees C Cryostar, and place immediately in a 37 degrees C water bath for 2 minutes.

   
   

2. Remove the cells from the vial and place in 10 ml wash media. This is necessary to remove traces of dimethyl sulfoxide from the cells.

   
   

3. Centrifuge cells for 10 minutes at 1200 rpm (no brake) at room temperature using the TJ-6 centrifuge.

   
   

4. Remove the supernatant above the cell pellet.

   
   

5. Resuspend the cell pellet in 7-10 ml of 1X Cyclosporin A media.

   
   

6. Aspirate half of culture media within 3-4 days. Add growth media and slightly increase volum by 5 ml. Increase the volume of media by 5-10 ml two times a week by aspirating off half of media from culture flask (do not to suction off cells from bottom of flask) and replacing it with fresh growth media. Cells can be harvested for extraction when a T-75 cm2 flask reaches a volume of 100 ml of media and there is a monolayer of cells on bottom of flask.

Solutions:

• Wash media: (1 liter)

  
  

 To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add: 10.0 ml 2.5 M (100X) Hepes buffer 1.2 ml 50 mg/ml gentamicin reagent  Filter sterilize through a 0.22 祄 cellulose acetate filter and store up to 2 weeks at 4 degrees C.

• Growth media: (1 liter)

  
  

 To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add:165.0 ml fetal bovine serum, heat inactivated at 50-60癈 for one half hour 12.0 ml 200mM (100 X) L-glutamine 1.2 ml 50 mg/ml gentamicin reagen  Filter sterilize through a 0.22 祄 cellulose acetate filter and store  up to 2 weeks at 4 degrees C.

• 1X Cyclosporin media: (100 ml)

 To 100 ml of growth media add:   1.0 ml 100X cyclosporin A

• 100X Cyclosporin A: (100ml)

  
  

 Dissolve 1 mg CSA in 0.1 ml ethanol in a sterile 15 ml centrifuge  tube with a small magnetic stirrer.  Add 0.02 ml (=  20 祃 ) of Tween  80 and mix well.  While continually stirring, add 1 ml  RPMI drop by  drop.  Bring to a final volume of 100 ml with RPMI.  Filter sterilize witha 0.22 祄 filter. Store at 4 degrees C for up to 4 months.

References:

Dr. Richard Todd, Psychiatry Department, Clinical Science Research Building.