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Protocol

Detection of Amphotrophic Retroviruses
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S+L Assay Sarcoma +, leukemia - virus
Day 0 Note: This section is to be completed in 1 hour. Do not vortex viral supernatants. 1. Make lx DEAE Dextran solution from stock lOx DEAE (200 ug/ml Dextran in HOH) using McCoy's medium. 2. Remove McCoy's medium from PG-4 dishes plated on day -1. 3. Add 0.5 ml lx DEAE Dextran solution; incubate for 30 min at 370, 5% C02- 4. Thaw viral samples. Make a 10-1. 10-20, 10-4 dilution of supernatant sample in 1 ml McCoy's medium. 5. Plate 0.5 ml diluted supernatant on respective dishes. Incubate for 2 hours at 370. 6. After 2 hrs., remove supernatant from dishes; add 2.5 ml McCoy's medium per dish. Day 3 Look for plaque formation on lOx without phase. NOTES: A) Polybrene (final 8 ug/ml) may be substituted for DEAE Dextran. Do viral dilutions in the media/dextran solution and infect cells with 1/2 ml of this infection media. During the infection period, rock the plates back and forth periodically. Due to the low volume, cells in the center of the plate are exposed to less media. B) The PG-4 cell line seems to be very sensitive to overgrowth and nutrient conditions as they are subject to morphologic changes due to stress in culture. When expanding with this cell line, split cells before or just at confluence. Do not let them become overcrowded and consistently change the media every few days.
References: Eckner, R.J., and Hettrick, K.L. (1977) Defective Friend spleen focus-forming virus: interfering properties and isolation free from standard leukemia-inducing helper virus. J. Virol. 24: 383-396. Miller, A.D., Law, M.F., Verma, I.M. (1985) Generation of a helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate resistant dihydrofolate reductase gene. MCB 5(3): 431-437.

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