Preparation of Virus Stocks

3.4.1. Making a Virus Stock From a Plaque

1. Set up one to two wells (24-well plate) of mouse 3T6 cells per purified mutant plaque so that they will be 75-80% confluent at the time of infection.

2. When the cells are ready, infect one or two wells per mutant by adding one-quarter to one-half of the purified plaque stock (see Note 15). Incubate at 37°C for 1 h and add approx 1 mL of prewarmed 1X DMEM containing 1% HICS. Monitor for CPE (see Note 14).

3. When the cells have significant CPE or have been infected for 10-14 d, harvest them by scraping the cells into the medium with a rubber policeman (see Note 16). Freeze-thaw the virus three times, then centrifuge 5 min at 2200g to remove the cellular debris. Store the virus at -70°C.

4. Using the newly obtained virus stocks and following the directions given above, infect one 35-mm plate of 75-80% confluent mouse 3T6 cells per mutant (see Note 17). Harvest as above and repeat using the new virus stock to infect the next larger plate of cells (35-mm^60-mm^100-mm^150-mm). Because the titer of the virus stocks is unknown, estimate the amount of virus needed. As a starting point, use approx 0.5-1 mL to infect 35-mm plates, 1 mL to infect 60-mm plates, 2 mL to infect 100-mm plates, and 3 mL to infect 150-mm plates. (Two 150-mm plates of cells will need to be infected if a large virus stock of greater than 200 mL is to be generated.)

3.4.2. Making a Large Virus Stock

1. Set up 10 150-mm plates of 3T6 cells per mutant so that they are 75-80% confluent at the time of infection.

2. Aspirate the medium from each plate and add approx 3 mL of mutant virus per plate. Rock the plates to ensure that all the cells are covered by the liquid. Incubate at 37°C for 1 h, then add 22 mL of warmed 1X DMEM containing 1% HICS. Monitor the cells for CPE and harvest when most of the cells are dead and detached from the plate. Harvest the virus by collecting the medium and spinning out the cells or by scraping the cells into the medium, freeze-thawing three times, and centrifuging to remove the cellular debris (see Note 16). Aliquot approx 1 mL of each mutant stock to a tube to use in a plaque assay. Store the remainder at -70°C in a large volume or as smaller aliquots for easy thawing upon use.

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