Determination of Retroviral Titer

1. On the day before the retroviral infection is to be done: Seed a 6-well tissue culture plate with 3T3 fibroblasts at a density of 2 x 104 cells/well.

2. On the day of infection add 100, 200, or 400 |L of the retroviral supernatant in a final volume of 2 mL GM to different duplicate wells. Add 8 |g/mL polybrene. The control well receives 2 mL of GM + 8 |g/mL polybrene.

3. Incubate in a humidified, 5-7% CO2 incubator for 2 d.

4. Fix and stain with X-gal as in Subheading 3.3.1.

5. Count the number of lacZ-positive cells in each well.

6. Plot volume vs number of lacZ-positive cells. The y-intercept yields the approximate number of infectious retroviral particles per microliter.

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