Using Fluid From Primary D. innubila Cells Infected with DiNV to Test Infection on Myd88 cells
Experimental Plan:
I want to use the aliquots of DiNV containing fluid from infected D. innubila cells as a test liquid on the Myd88 cells I have, they are on passage 20. But I have a limited supply of the fluid, so I have to be careful about the design. My idea is to use Dv-1 cells as a control because I don’t know how this fluid causes infection in Dv-1 cells, it hasn’t been used on them. I also know I have to take day 0 flasks for samples of base level of virus just after adding it.
Flasks needed:
3 Myd88 flasks, 1 for day 0 and 2 for day 5
3 Dv-1 flasks, 1 for day 0 and 2 for day 5
I want to sample the flasks after 5 days because that is when the replication of DiNV is the most in Dv-1 cells. I will want to take supernatant and cell fractions of the flasks for qPCR analysis.
20230106 Plating Cells
- First I need to plate the cells into the flasks I need, and I want to try to do it in a quantified way. The main issue is that the Myd88 cells do not replicate as fast as the Dv-1 cells do
- I decided to make 4 flasks of each line
- I read on this website that I should plate 2,000-5,000 cells per cm^2 (this might have been too many but we’ll see) which comes out to 50,000-100,000 cells per 25cm^2 flask (this may have been fine, this other resource suggests using 700,000 per flask)
- I decided to try 50,000 cells for the Dv-1 cells and 75,000 cells for the Myd88 cells per flask
- I scraped up Myd88 cells and counted them with a hemocytometer
| section | quadrant 1 | quadrant 2 | quadrant 3 | quadrant 4 | section average |
|---|---|---|---|---|---|
| 1 | 242 | 221 | 227 | 211 | 225 |
| 2 | 272 | 281 | 203 | 223 | 245 |
- Each section was averaged, and the average of the two sections was taken
- Total Myd88 average: 235
- The cells per mL is the average * 10^4
- 235 * 10^4 = 2,350,000 cells per mL
- If I want 75,000 cells per flask, then I want 300,000 cells total (4 flasks)
- (2,350,000 cells)(5mL) = (300,000 cells)(xmL)
- x = 40mL
- I then divided these by 3 to get the amount of cells in ~20mL (the amount needed to make 4 flasks) and so I used 2mL of cells and 20mL of medium to dilute the cells
- Dv-1 cells were also counted with the hemocytometer
| section | quadrant 1 | quadrant 2 | quadrant 3 | quadrant 4 | section average |
|---|---|---|---|---|---|
| 1 | 216 | 239 | 341 | 299 | 274 |
| 2 | 305 | 328 | 345 | 295 |
- Each section was averaged, and the average of the two sections was taken
- Total Dv-1 average: 296
- The cells per mL is the average * 10^4
- 296 * 10^4 = 2,960,000 cells per mL
- If I want 50,000 cells per flask, then I want 200,000 cells total (4 flasks)
- (2,960,000 cells)(5mL) = (200,000 cells)(xmL)
- x = 74mL
- I then divided these by 4 to get the amount of cells in ~20mL (the amount needed to make 4 flasks) and so I used 1.2mL of cells and 20mL of medium to dilute the cells
- Flasks were placed in the 22C incubator to grow over the weekend
20230109 Infecting Cells
- Thawed 6 day 26 innubila primary fluid infected with DiNV on ice (later I noticed that one of these had less than 40ul, so I thawed a 7th one in my hands)
- Spun down aliquots
- Got 5mL tubes for the day 0 supernatant freezings
- Added 40ul DiNV fluid to each of 6 flasks made on Friday (see above). 3 were Dv-1 and 3 were Myd88
- For 1 Myd88 and 1 Dv-1 flask I aspirated the supernatant after adding the virus and put it in a 5mL tube
- I then took the 5mL tube and the flask it came from and placed them in the -80
- These were labeled “day 0” for initial samples
- The others were rocked back and forth gently after virus addition, then laid horizontal
- I ethanol sprayed two old p1000 pipette tip boxes and used them as boxes for the infected flasks to keep them separate in the incubator
- Put the flasks in the 22 degree incubator until Saturday where I will harvest them
- Sprayed everything with ethanol after this to make sure any virus residue had been eliminated
20230114 Freezing Cells
- Started up cell culture hood
- I had 6 flasks to separate and freeze: 2 infected flasks per cell type and 1 non-infected flask per cell type
- For every flask I removed ~5mL of supernatant from the flasks and placed in a 5mL sterile tube, these were labeled A and B for the replicates so I could tell which flask they came from
- The flasks were also labeled
- All flasks and 5mL tubes of supernatant were frozen at -80 C
Information on the samples is here, and the metadata is here.