Protocols

End-point Dilution Assay

The end-point dilution assay (EPDA) is useful for a variety of assays. A 96-well plate EPDA may be used to replace the plaque assay and plaque purification as a method for either determining viral titer or identifying and purifying recombinant virus. A modified 12-well plate EPDA can be used as a routine method for determining viral titer; it is useful for estimating the efficiency of the initial co-transfection, identifying infected cells, approximating viral titers, and amplifying viral titer. In the 12-well EPDA, individual wells containing equal amounts of insect cells are inoculated with 100, 10, 1 or 0 µl aliquots of the original transfection supernatant, wild-type virus, or recombinant XylE positive control viral supernatant. A visual comparison between cells in wells inoculated with 100, 10, 1 and 0 µl is used to estimate the viral titer.

For example, if cells receiving 100 µl of the initial co-transfection supernatant look infected in the EPDA, but cells receiving 10, 1 and 0 µl do not, then it is likely that the viral titer is low and should be amplified to produce a high titer stock. If wells receiving 100 µl of the original co-transfection supernatant look similar to those receiving 0 µl, it is likely that the original co-transfection did not result in a significant viral titer and must be repeated. When assaying the efficiency of a co-transfection or estimating the titer of a virus stock, if the EPDA shows a 10 fold decrease in the number of infected cells between dilutions, amplify the virus once or twice more to generate a high titer stock for protein production. However, if all three wells (100, 10, 1 µl) show equal signs of infection, the viral titer is high, ~ 2 x 10⁸ plaque-forming units (pfu) /ml. High titer recombinant virus stocks are used for infection of cells at optimal multiplicity of infection (MOI = # of virus / # of cells) resulting in maximum protein production.

If the EPDA is used as an amplification step to generate a high titer stock, cross contamination between wells containing different viruses, eg., the highly infectious wild-type virus used as a positive control, can be avoided by using separate 12 well plates.

EPDA controls are recommended. The recombinant virus from a pVL1392-XylE transfection is a particularly useful positive control. Infected cells producing the the XylE protein turn yellow in the presence of catechol and are easily identifiable.

Protocol

1. Dilute log-phase Sf9 cells (with greater than 98% viability) to 1 x 10⁵ cells /ml with fresh TNM-FH medium. Seed 1 x 10⁵ Sf9 cells per well on a 12-well plate (BD Falcon™, Cat. No. 353043). Allow cells to attach firmly, approximately 10 min. Confirm 30% confluency by visualization on a light microscope. Replace medium with 1 ml fresh TNM-FH.
2. Add 100, 10, 1 and 0 µl of the recombinant virus supernatant obtained 5 days after the start of co-transfection (or other virus stock), to separate wells. Do the same for the positive control, e.g., pVL1392-XylE supernatant.
3. Incubate the cells at 27°C for three days. Examine the cells for signs of infection.
4. A successful transfection should result in uniformly large infected cells in the 100, 10, and 1 µl experimental wells. The cells in the 0 µl control wells should not look infected because they were not inoculated with virus.
5. If only the 100 µl and 10 µl wells seem to have infected cells and the 1 µl well looks more like the control, the titer of your virus supernatant is low. Amplify the virus an additional time before proceeding with protein production.

• Protein production can be analyzed by western blot analysis (if antibodies are available) or by Coomassie blue-stained SDS-PAGE gel by harvesting cells from the 100 µl well and lysing in appropriate lysing buffer.
• The virus supernatant from the 100 µl well may be kept as the first viral amplification stock, however care should be taken to avoid cross-contamination between wells containing different virus.
• To further purify the virus population, a plaque assay purification of the co-transfection supernatant may be performed using the approximate titer obtained from the EPDA.