May 31, 1990
Rosalie Veile
Purpose:
Blood Samples can be stored frozen as a backup in case an LCL is needed at a later date.
Time required:
15 minutes to freeze 1-4 cryotubes placing them directly into the -135 degrees C freezer; orone hour to freeze the tubes using the Cryomed freezing chamber. Cells have been shown to be more viable if temperature is lowered gradually with the freezing chamber.
Procedure:
Freezing cells:
Pipette 1.0 ml of whole blood into 2 cryotubes (1.25ml).
Add to each cryotube 100 µl dimethyl sulfoxide (DMSO), or 10% of the volume of blood.
Immediately begin freezing the whole blood in the cryomed freezing chamber until the chart drive printer reads -90 degrees C.
Quickly transfer the frozen sample to long term storage in a -135 degrees C freezer or a liquid nitrogen storage container. These whole blood samples have been shown to be viable for as long as 5 months by G. Chenevix-Trench, et al.
Thawing cells for transformation:
When an LCL is needed, the cells are thawed rapidly in a 37 degrees C water bath: Place the cryotubes in a bubble rack. Shake the rack to help thaw the cells, usually for 1-2 minutes.
With a 1 ml disposable pipet, transfer the sample to a 15 ml conical centrifuge tube filled with 10 ml of wash media.
Centrifuge the cells for 10 minutes at 1200 rpm, no brake, at room temperature.
Aspirate the wash media to just above the cell pellet. Wash the pellet again with 10 ml wash media and centrifuge as in step 7. Repeat the wash a total of 4 times or until the red cell contamination is minimal. If the red cell contamination is not eliminated, several days in culture will decrease the amount of red cells substantially.
Aspirate the wash media and resuspend the cell pellet in 300 µl filtered supernatant from a B95-8 marmoset culture containing Epstein Barr virus. Transfer the cell suspension to a T-25cm2 flask and incubate for 2 hours at 37 degrees C with 5% CO2. If there is a very small volume of cells, leave the cells in the 15 ml centrifuge tube for the incubation.
After incubation, add 800 µl RPMI-1640 containing 20% fetal bovine serum and 2X Cyclosporin A.
Using a 5 ml disposable pipet, plate out cells in serial dilution in a 96-well microtiter plate: Transfer half of the cells in the first well into a second well. Add enough media to fill the second well. Then take half of this cell/media mixture and transfer to a third well. Fill up the third well with media. Incubate at 37 degrees C with 5% CO2.
Feed the cells twice-weekly by removing half of the old media and replacing with fresh media until transformed colonies are apparent (ususally 2-3 weeks). The new media should contain 1X CSA.
Subculture cells to a 24-well plate before transferring to culture flasks. Maintain the subcultures on growth media (no CSA).
Solutions:
Growth media: (600 ml)
To 500 ml of sterile RPMI 1640 with 2 mM L-glutamine, add: 90.0 ml FBS 6.0 ml 200 mM (100X) L-glutamine 0.6 ml 50 mg/ml gentamicin reagent Note: Filter sterilize through a 0.22 µm filter and store up to 2 weeks at 4 degrees C.
Wash media: (1 liter)
To 1 liter of sterile RPMI 1640 with 2 mM L-glutamine, add: 10.0 ml 2.5 M (100X) hepes buffer 1.2 ml 50 mg/ml gentamicin reagent Note: Filter sterilize through a 0.22 µm filter and store up to 2 weeks at 4 degrees C.
2X Cyclosporin A media: (1 µg/ml)
To 100 ml of growth media add 2 ml of 100X Cyclosporin A.
100X Cyclosporin A: (100 ml)
Dissolve 1 mg CSA in 0.1 ml ethanol, add 0.02 ml Tween 80 and mix well. While continually stirring, add 1 ml RPMI, drop by drop. Quanitate to a final volume of 100 ml with RPMI. Filter sterilize and store at 4 degrees C for up to 4 months.
References:
Chenevix-Trench, G., et al, Am. J. Hum. Genet. 46:635-636, 1990.
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