Plan for Diluting DiNV to Certain FFU Concentrations for Injection

Kent and I’s work on developing a IFA based assay for viral titering (see here) has yielded a possibly good method for determining FFU (focus forming units) of our DiNV fluid from cell culture.

The one fluid we have tested so far (was 14 Cq) yielded: 210,000 FFU/mL (210 FFU/ul) or 267,500 FFU/mL (267.5 FFU/ul). This was two times where I counted the same well in the same plate (10^-3 for 48hr Dv-1 cells) and got slightly different counts, but Dr. Orozco said those counts were well within the margin of error to be worried about.

Based on those numbers, the average is 238.75FFU/ul, and is what I’ll be basing my calculations off of.

For all of my infections that now use the nanoinjector, I have been using 27.6nl as the volume. I think I don’t want to change that volume because it has been working well. Unfortunately I have not tested the fluid we titered in flies yet, so I will have to test an undiluted version in these infections. If I use the undiluted virus at 238.75FFU/ul, and 1ul is 1000 nano liters (nl), then when delivering with this virus and volume I would give each fly 0.23875 FFU/nl. If we multiply this by 27.6nl, that is 6.375FFU per injection. This seems kinda small, but FFU doesn’t actually mean viral particles, and it is probably to be expected that DiNV doesn’t do as good of a job infecting Dv-1 cells as it would D. innubila cells. The FFU is just a way to standardize the amount of virus going into experiments.

What I would like to do after the undiluted stock, is to take some dilutions. I think the best way to do that is to consider a desired total FFU I want to give each fly based on the 27.6nl I want to use.

  • So if I want to give each fly 6FFU per injection, I should dilute the stock virus to : (6/27.6) = 0.217FFU/nl
  • 3FFU per injection: 3/27.6 = 0.1086FFU/nl
  • 1FFU per injection: 1/27.5 = 0.0363FFU/nl
  • 0.1FFU per injection: 0.1/27.6 = 0.00362FFU/nl
  • 0.01FFU per injection: 0.01/27.6 = 0.000362FFU/nl

That’s 7 conditions I’d need to test: undiluted, 6FFU, 3FFU, 1FFU, 0.1FFU, 0.01FFU, and a control. However do I need to do a different control to compare with each dilution? Because I could dilute cell culture medium the same way I dilute the viral fluid. Also what is the best way to do these dilutions, in water, viral buffer, 1X PBS? I think maybe the best thing to dilute in is cell culture medium, then the control is good for all of them.

Ideally I’d want like 40-50 flies in each of these conditions (or more), but that is becoming a lot of work. That would be like 300+ flies. I think I would also first try this out on males? Or should I do both sexes?

10 male and 10 female control
10 male and 10 female undiluted
10 male and 10 female 6FFU
10 male and 10 female 3FFU
10 male and 10 female 1FFU
10 male and 10 female 0.1FFU
10 male and 10 female 0.01FFU

That is 140 flies in one experiment. My guess is that I can definitely infect that many flies in less than 2 hours (if we are not counting set up time with the needle and refill time, which will add more time).

If I repeated that experiment 3 times, that would be 30 flies in each group, not great replication but not nothing either.