Testing the Nanoject II Injector to use for Virus Fly Infections
Potentially something that will make our infections more reproducible and comparable would be to use the injector needle to deliver a set dose of fluid to flies for infections. And if we get a better way to titer DiNV, we could potentially calculate the number of viral particles per does given to each fly. But no one has use this injector in the lab so I had to figure out how to use it.
I tested the injector on 40 D. innubila of both sexes, around 7 days post emergence in age. I injected them with sterile cell culture medium. At first I started with 69nl of fluid, which is the maximum, but then I went down to 27.6nl. This lower volume seemed to work better, but more testing needs to happen before I can be sure which volume is best.
This process will be tested a few more times and a full protocol will be written. Here is a consolodated description of what I did to get the injections working.
Pulling Needles
- Use Lundquist needle puller in 5057 (I was shown how to do this by CJ)
- Make sure that the numbers are set to 95 for Heat 1 and 78 for Heat 2
- Only use the needles provided with the injector (not the ones by the puller)
- Make a box (freezer tube box) without the tube holder in it, with a cylinder of sticky tack on it. This will be where you place the needles so they are secure but don’t touch anything with the point after you make them
- Stick the capillary tube into the holders, making so it is halfway on each of the holders
- Make sure it is vertical, in the groove in the holder, then screw in each holder tight
- Close the door, turn on the machine, and press pull
- Turn off the machine inbetween
- Wait a second to take out the needle because it can be hot
- Gently take out the two needles and place in the box
- Re-do to make other needles
Steps Done During Testing that Worked
- Made up at 0.65mL tube with 100ul of sterile cell culture medium, 10% FBS
- Used all the sterile materials like doing a real virus infection
- Note I will have to come up with a protocol for sterilizing the nanoject
- Setting up the injector
- Make sure injector is in home position (metal stick is flush with collet when slightly unscrewed)
- Take off collet and first o-ring
- Push a capillary tube through the o-ring a few times to make sure it is open, this ring is almost too narrow, and can make it super hard to put the needle into the machine
- Once it is sufficiently loose, put it back together
- Look at the needle tip under the microscope and use a forceps to cut off the tip of the needle, make sure not to take off too much and make it too wide. Without doing this the needle won’t suck up fluid
- Backfilling the needle
- Without a needle, use a 5mL syringe to suck up a small amount of mineral oil
- Flick to dislodge bubbles and use the plunger to release air bubbles
- Wipe up any excess oil with a kimwipe
- Add the metal needle (from injector box) to the syringe
- Very gently press plunger until droplets of oil come out. Do this until confident there are no air bubbles in the needle
- Take the pulled glass needle (with the clipped tip) and insert the metal needle into it almost to the tip. Slowly add oil to the glass needle, pulling back the metal needle to fill it with oil without making bubbles, fill it to the end
- Put the glass needle into the holder, it will fit around the metal inner part of the injector, through the collet, and you have to push it through the first o-ring. This can be tight so you have to gently but firmly push down to get it to seat through the o-ring
- Screw the collet tight
- Filling the needle
- Press and hold the eject button until you hear two beeps and it stops (long time), hold the needle over a kim wipe because the excess oil will come out of the tip
- Then set up your injection solution in one of the colorful racks that can fit 4 50mL conicals, so that you can see the contents of the tube in the rack. Set it up high, on the microscope and on top of the CO2 so you can see easily horizontally into the tube
- Put the glass needle in the tube so that it is submerged
- Press and hold fill on the injector and hold the needle in the solution it needs to suck up. Hold the button and needle until the machine beeps
- Look at the needle end under the microscope on a kim wipe. Press eject a few times until the liquid starts to bead out of the tip. This can take a few times and then all the liquid will come out at once
- Follow the process very similar to the DiNV infection protocol for metal needles
- Each time you poke a fly, you you press inject with the machine
For this test, I tested 40 flies, and made 4 vials. The first two had more time on CO2 than the others, actually the last two vials had only 6-7 min on CO2. It is really the setup that takes a long time, the injections are pretty similar to the metal needle method.
I used a different needle for the 2nd two vials than the first two.
The flies tested:
| tube | volume | time | N# | day1 | day2 | day3 | day4 | day5 | day6 | day7 | day8 | day9 | day10 | day11 | day12 | day13 | day14 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 69-27.6nl | ~10+ min | 10 | NA | NA | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 9 | |
| 2 | 27.6nl | ~10+ min | 10 | NA | NA | 8 | 8 | 8 | 7 | 7 | 7 | 7 | 6 | 6 | 6 | 6 | |
| 3 | 27.6nl | 6 min | 10 | NA | NA | 10 | 10 | 10 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | |
| 4 | 27.6nl | 7 min | 10 | NA | NA | 10 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |