Inoculating 48 Plates of Dinn and Dv-1 Cells with a Serial Dilution of DiNV to Test Virus Titering Methods
Kent and I are going to try doing both endpoint and focus forming unit assays on serial dilutions of DiNV in both Dinn cells and Dv-1 cells. For this Kent had plated 4 48 well plates of Dinn cells and 4 48 well plates of Dv-1 cells last week, and let them grow for about a week. We will both get 4 plates, two of each cell type, and we will fix them at 24 hours and 48 hours post inoculation.
Each plate has the same layout, where 4 wells per column have cells, except for the first and last wells. All plates will get the same dilution layout, see below:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
|---|---|---|---|---|---|---|---|---|
| A | cell control | 10^-2 | 10^-3 | 10^-4 | 10^-5 | 10^-6 | ||
| B | 200ul medium | 200ul 10-2 dilution | 200ul 10-3 dilution | 200ul 10-4 dilution | 200ul 10-5 dilution | 200ul 10-6 dilution | ||
| C | 200ul medium | 200ul 10-2 dilution | 200ul 10-3 dilution | 200ul 10-4 dilution | 200ul 10-5 dilution | 200ul 10-6 dilution | ||
| D | 200ul medium | 200ul 10-2 dilution | 200ul 10-3 dilution | 200ul 10-4 dilution | 200ul 10-5 dilution | 200ul 10-6 dilution | ||
| E | 200ul medium | 200ul 10-2 dilution | 200ul 10-3 dilution | 200ul 10-4 dilution | 200ul 10-5 dilution | 200ul 10-6 dilution | ||
| F |
I calculated exactly how much medium and volume of each dilution is needed. We are not using the 10^-1 dilution, so I only made a small amount of that, 400ul are needed for the next dilution, so there is 100ul excess. The other dilutions have enough volume for what is needed for each well (200ul in 16 wells, so 3.2mL needed), and what is needed for the next dilution (400ul). Because for each of these dilutions I made 4mL total, there is 200ul excess for each dilution.
| 10^-1 | 10^-2 | 10^-3 | 10^-4 | 10^-5 | 10^-6 |
|---|---|---|---|---|---|
| 50ul of non-diluted virus solution | 400ul of 10^-1 virus solution | 400ul of 10^-2 virus solution | 400ul of 10^-3 virus solution | 400ul of 10^-4 virus solution | 400ul of 10^-5 virus solution |
| 450ul of medium | 3,600ul of medium | 3,600ul of medium | 3,600ul of medium | 3,600ul of medium | 3,600ul of medium |
| total volume 500ul | total volume 4mL | total volume 4mL | total volume 4mL | total volume 4mL | total volume 4mL |
- All dilutions and processes were done in the cell culture hood with sterile technique and filter tips used
- The virus we used as the stock was from DiNV passage 4 from 08072023. This had a qPCR Cq value of 14
- All medium used in the dilutions was serum free medium, so the medium made was 49.5ul of Schneider’s Drosophila medium and 50ul of Gentamicin
- The serial dilutions were made in 15mL conicals, except for the 10^-1 which was made in a sterile cryo tube
- The virus solution was thawed on ice, and vortexed before use
- 450ul of medium and 50ul of virus solution were added to the cryotube and vortexed
- 5 15mL tubes were filled with 3.6mL of medium
- 400ul of the 10^-1 diltuion was added to the first 15mL tube of medium and vortexed
- The dilutions preceded the same way, the first dilution was vortexed, and 400ul of it was added to the next dilution
- The final 15mL tube was left without any virus
- For each plate, the medium already in the wells was removed by dumping the plate on a glass dish filled with paper towels, then blotting the dish on autoclaved paper towels (blot means tap down hard on the towels)
- I only did my 4 plates, Kent did his. He did his dilutions in 5mL volume, similar concept of how he diluted
- I added 200ul of plain medium to the 2nd column of all plates
- I added 200ul of the 10^-2 diltuion to the 3rd column of all plates
- I added 200ul of the 10^-3 diltuion to the 4th column of all plates
- I added 200ul of the 10^-4 diltuion to the 5th column of all plates
- I added 200ul of the 10^-5 diltuion to the 6th column of all plates
- I added 200ul of the 10^-6 diltuion to the 7th column of all plates
- I only added these to the 4 wells that have cells
- 200ul of molecular grade water was added to all of the surrounding wells to prevent evaporation
- The plates were put in the 23C incubator for either 24 or 48 hours
More information on the plate dilutions can be found here