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Method: Lymphocyte Transformation

May 30, 1990

Rosalie Veile


Principle:

Special reagents:

Time required:

Procedure:

  1. Collect 27 ml of anticoagulated blood in 3 yellow top tubes (citrate), 9 ml each. The blood should be set up in culture as soon as possible for best results. Blood should be kept at room temperature prior to use in this procedure.

  2. Wipe the exterior of the tubes of blood with EtOH, divide evenly and transfer the blood into 2-50 ml tubes. Bring the volume of each tube up to 40 ml with wash media

  3. Place 10 ml of histopaque -1077 into two other 50 ml tubes. Overlay the blood and wash media mixture onto the 10 ml of histopaque. Do this very slowly making sure not to mix the two layers. See figure 1, below.

  4. Centrifuge tubes for 30 minutes at 1500 rpm at room temperature (no brake), in the TJ-6 centrifuge. Aspirate the top layer down to within 1/4" of the white blood cell layer.

  5. Collect the WBC layer using a 10 ml pipet, moving the pipet in a circular motion around the inside of the tube just below the surface of the WBC layer. Transfer the WBC layer to another 50 ml tube. See figure 2, below.
  6. Bring the volume of each tube up to 50 ml with wash media, gently invert tubes to mix.

  7. Centrifuge the tubes for 20 minutes at 1200 rpm at room temperature (no brake) in the TJ-6 centrifuge. Aspirate supernatant.
  8. Add 12 ml wash media, resuspend the cell pellet and transfer to 15 ml centrifuge tube.

  9. Centrifuge 8 minutes at 1000 rpm at room temperature (no brake) in the TJ-6 centrifuge. Aspirate supernatant.

  10. Cell counts can be done to determine the appropriate volume of media to be added to the cells. Cells should be set up in culture using a minimum of 2.6 x 106 cells/ml and not more than 7 ml per 25cm2 flask. The average WBC count of whole blood ranges from 1 x 106 cells/ml to 3 x 106 cells/ml. An ideal primary culture should contain between 5 and 7 ml of cells/25cm2 flask. Cells are resuspended in 10 ml of RPMI (without serum) for counting. If cell counts cannot be done, set up cultures in 5-7 ml of fresh RPMI, 20% FBS, and 2 g/ml cyclosporin A.
  11. Inoculate cells with an equal volume of virus (refer to procedure describing the maintainence of the B95-8 cell line). Incubate cells at 37 degrees C with 5% CO2 and loose caps on flasks.

  12. Feed the culture after 24-48 hours if the media has turned yellow. This is done by removing 1/2 of the media and replacing it with 1 g/ml cyclosporin A media. Discontinue cyclosporin A after 3-4 weeks. Do not overfeed cells. Do not increase the volume of media for at least 2 weeks. If cells do not seem to be growing, reduce the volume of media and feed only once a week. After 2 weeks, when cells are very clumpy and the media is changing color from orange to yellow within 3 days incubation, increase the volume of media by 10-20 ml. Cultures are always fed by removing 1/2 of the old media and replacing it with a slightly increased volume of fresh media. Cultures can be split when they reach 25-30 ml of media in a 25 cm2 flask.
Solutions:

References:

Sigma Diagnostics Histopaque catalog, July 1987.

Dr. Dilley, Department of Psychiatry, Washington University Medical Center.