Testing Concentration of D. innubila Primary Fluid Infected with DiNV using Ultrafiltration Centrifugation
I found a few papers suggesting that you can use ultrafiltration centrifugation to concentrate viral particles in cell culture supernatant. I also found that we have some of these ultrafiltration tubes in the lab, 15mL tubes from Amicon with a 3,000 kDa molecular weight cut off (MWCO), and 15mL tubes from Sartorius with a 10,000 kDa MWCO. I had orientally wanted to used 100,000 kDa MWCO tubes because DiNV should definitely be larger than 100,000 kDa, and these filters remove anything lower than 100,000 kDa. But I decided to just use what we have and try it out. I used the Sartorius tubes for this test because it has the highest MWCO.
I used this document about Amicon filters and this Amicon user’s protocol to sort of come up with a protocol. I now worry that I used too fast of a spin speed, but that is what is recommends on the Sigma page. I used the Sigma page to come up with the spin speed and time. I used the protocol to try a wash step of the filter paper (I didn’t do it the same way though).
Preparing samples
- All sample information can be found here
- I had 5mL of supernatant fluid from the flask of innubila cells (presumably producing DiNV) frozen at -80 that I took on 20220222
- I took another 5mL supernatant fluid today and re-fed the flask with 5mL o f 20% FBS with 4% mushroom extract and gentaminicin
- The frozen sample was thawed on ice
- I aliquioted out 50ul, 100ul, and 1mL of each sample, 50ul for the DNA extraction, 100ul for backup for the DNA extraction, and the ~1mL was sort of the left over
- Below are all the samples taken, including sample tubes made after concentration
| sample_ID | date_collected | sample_type | volume_ul |
|---|---|---|---|
| 1 | 20230222 | non-concentrated | 50 |
| 2 | 20230222 | non-concentrated | 100 |
| 3 | 20230222 | non-concentrated | 1000 |
| 4 | 20230222 | concentrated | 50 |
| 5 | 20230222 | concentrated | 700 |
| 6 | 20230222 | flow-through | 50 |
| 7 | 20230222 | flow-through | 1000 |
| 8 | 20230213 | non-concentrated | 50 |
| 9 | 20230213 | non-concentrated | 100 |
| 10 | 20230213 | non-concentrated | 1000 |
| 11 | 20230213 | concentrated | 50 |
| 12 | 20230213 | concentrated | 700 |
| 13 | 20230213 | flow-through | 50 |
| 14 | 20230213 | flow-through | 1000 |
| 15 | 20230222 | wash | 450 |
| 16 | 20230213 | wash | 450 |
| 17 | 20230222 | wash | 50 |
| 18 | 20230213 | wash | 50 |
- 4mL of fluid from each collection would be put into the ultrafiltration tubes to be concentrated, there was one per collection date
- I used the centrifuge in 5055, I set it to 4C and let it run for 10 minutes so it would cool down
- I had no idea how long to centrifuge because I wasn’t sure how fast the liquid would pass through the filter. Based off of this document about Amicon filters I was thinking about 30 min, but the MWCO is different
- I started with a 2 min spin a 1,500g at 4C, then I kept adding 5 min, 6 min, 5 min, and another 2 min, resulting in a final spin time of 20 min
- It looked to me that the 4mL had concentrated down into something a little less than 1mL. I thought this might be a good place to start
- Everything was kept on ice or at 4C the entire time, I brought the filter tubes back to the lab
- I pipette mixed the liquid in the filter reservoir of the ultrafiltration tubes and put it into a new tube per collection day. There was about 700ul in volume
- Aliquioted 50ul of that into separate tubes to be used for DNA extraction
- I took 1mL and 50ul from the flow through liquid per sample as well so I could use them to check for viral DNA that could have passed through the filter
- And I used 500ul of 1X PBS to “rinse or wash” the filter by pipetting over and over to get any extra virus left on the filter, and kept those as sample as well
- Every sample was frozen at -80 C