Fixing and Staining Dinn and Dv-1 Cells That Have a Serial Dilution of DiNV to Test Virus Titering Methods

Using the plates generated here. Using acetone to fix the plates at either 24 or 48 hours post inoculation. Then we use the polyclonal nucleocapsid antibody from the rabbit final bleed 13345 to recognize the virus. And we have to also add the conjugate, a goat anti-rabbit antibody with alexa fluor 488 which will glow green like GFP. That will bind the first antibody, so we can see the nucleocapsid in a microscope with fluorescent capabilities.

Fixing 24 hour plates 20230908

  • Kent prepared fresh 80% acetone (using molecular grade water as diluent) and placed it in the -20 freezer to get to -20 degrees
  • Each of us took 2 plates, one from each cell type to fix for 24 hours
  • The plates had their fluid dumped off into the glass dish with paper towels, and tapped down on fresh paper towels (note that these towels contain virus and were disposed of the in the autoclave trash)
  • Then we took a 50mL serological pipette and filled with with the cold 80% acetone, this was dripped into each well that had cells in the two plates, just until the wells were full. We used the slow setting on the pipette to avoid disturbing the cells too much
  • Placed the plates in the -20 freezer for 30 minutes to fix
  • After the 30 minutes, the acetone was dumped off and taped out in the paper towels
  • The plates air dried out in the open for 20 minutes
  • Then the plate were placed in the -20 for storage until staining

Note that because it was a Saturday for 48 hours, Kent came in and did the fixing for both of our plates

20230912 Antibody Staining Cells

  • Took plates out and warmed them to room temp in the hood
  • Warmed blocking buffer in the hood as well to room temp
  • All staining took place at Kent’s bench through, cells are fixed and can’t get contaminated by the air
  • Blocking buffer is:
    • DPBS
    • Glycine
    • 1% BSA (comes from powder fraction 5 from Dr. Ackley)
    • 0.1% Tween
    • This has been sterile filtered
  • Dripped blocking buffer on each cell well with the slow setting of the pipetter to cover each well. Blocking buffer helps to prevent extra binding of the antibody on things not specific
  • The plates were covered and let incubate with the buffer at room temp for 30 minutes
  • During this time we prepared the antibody
    • The antibody is final bleed 13345
    • Kent has aliquots of this in cryotubes with 500ul in each in the -20
    • When you want to work with one, you thaw it and add 50ul of glycerol
    • This makes it so that your antibody doesn’t freeze thaw when you keep it in the -20, and only that one tube should have the glycerol and be your working stock
    • We are using the 50% glycerol opened vial of nucleocapsid antibody
  • We want a 1:1000 dilution of antibody (if you think about the antibody already halved in the glycerol, it is kinda a 1:2000 dilution). Kent knows this dilution will work so we should use it
  • We are preparing 100mL of the 1:1000 dilution (there are 24 wells per plate, and 8 plates, meaning 192 wells. If we are adding 500ul of antibody to each well, that is 192 * 500 = 96,000ul or we can round to 100mL)
    • 100mL of DPBS with 0.1% Tween and 1% BSA into a sterile flask
    • 100ul of primary antibody
    • Mix/swirl solution
    • This solution should also be at room temp when used
  • After the 30 minutes in the blocking buffer, that was dumped into the sink and the plates tapped on paper towels
  • 500ul of the 1:1000 antibody solution was added carefully to each well with cells in the plates individually with a pipette
  • The plates were incubated in the cabinet (dark) for 1 hour with the antibody
  • After the incubation, the fluid was dumped into the sink and the plates tapped on paper towels
  • Each well was washed with DPBS by dripping into each well with the serological 50mL pipette to fill the wells
  • The plates let sit in the wash for 10 minutes
  • After the 10 minutes, the fluid was dumped into the sink and the plates tapped on paper towels
  • The wash with DPBS was repeated 2 more times for a total of 3 washes with DPBS
  • During the washes, we prepared the conjugate
  • We want to use the conjugate at 1:4000. With 192 wells, and 200ul of conjugate needed, 38,400ul total are needed, and we can round to 40mL. 40mL divided by 4000 is 10ul. 10ul of conjugate is needed
    • In a 50mL conical, 40mL of DPBS 1% BSA (no tween) was added
    • 10ul of Alexa Fluor 488 conjugate added
    • Invert to mix
    • Keep in drawer or cabinet out of light until used
  • After the 3rd wash, the plate is empty
  • Added 200ul of diluted conjugate (with the light off) to each well with cells, individually pipetting each well
  • The plates were covered and kept in the cabinet for 1 hour. The conjugate should not see direct light
  • After 1 hour incubation, the plates were washed 3 times again with DPBS as above (Kent did my plates for this as I was in class)
  • For the last addition of DPBS, we did not dump it off and left it in the plates to keep them moist

Looking at/Counting Plates

There is not much to say here, we were able to look at the plates in the new scope in Dr. Ackley’s lab. Counting and scoring these were hard/impossible. Images I took from the plates can be found here, but they are not exhaustive, and the way the images come out is different than how it looks through the eyepiece.

Firstly, we could see staining at 24 hours, which is good to know. It is in both cell types, and you are better able to see it in the 48 hour plate. The things to note is that there is a lot of background signal in the Dinn cells, from clumps or debris. This makes it tough to be confident what is virus or not. The other thing is that the whole cells do not light up, what we see are these pinpriks of glowing, which we think is the puncta that form during infection (although we are not sure they form these this early on in infection). This causes us problems because it is hard to tell what we confidently think is virus in the Dinn cells because there is other glowing cells. The other issue is how small the fluorescence is, so it can be hard to see/tell in the Dv-1 cells as well, even though it is more defined in those cells and has less background. What I obvserved is that it is really easy to see the virus in the 10^-2 wells, but there is too much to count. You maybe can count in the 10^-3 wells, but in the Dv-1 cells only. The further dilutions, it becomes hard to decern what is virus and what is not. I didn’t feel confident in the Dinn cells to do any counting of virus because I felt like I couldn’t decern what is virus versus what is other.

I counted the 10^-3 4 wells in the Dv-1 48 hour plate to the best of my ability:

well count1 count2
A 21 35
B 55 64
C 51 62
D 41 53

The count 2 was done on the 14th, two days later. We looked at the plate again that day. First we tried looking at them with Dr. Davido’s scope which has a 20X objective, but the glow was extremely faint and it was super hard to see anything. We think Dr. Ackley’s score has a photomultiplier that makes viewing these much more possible. So we did not count with Davido’s scope really. We looked at Dr. Ackley’s scope again and I re-counted the same dilution plate. It seemed easier than the first time, but still kind of frustrating to really know what was virus and what was some small background glowing.

For those numbers, I scanned across the well and used a counter to count how many virus positive dots I saw. A dot meaning just visible virus. It is a little hard to be sure you are not duplicating or missing something. This is for the “focus forming unit” calculation.

I did not really score any of the plates for end point titration. At the lower dilutions I got increasingly confused at whether I saw a puncta or not, even just 1, in a well. And that would make the well positive for virus. So I didn’t feel like I could accurately say is a well was negative.

If we average each day I did the counts for the 10^-3, that is either 42 or 53.5.
The equation for FFU/mL is : ((FFU# *(1000ul/ul of virus added to well))/dilution factor)

  • So this would be:
    • ((42 * 1000ul/200ul)/10^-3) = 210,000 FFU/mL or 210 FFU/ul ?
    • or ((53.5 * 1000ul/200ul)/10^-3) = 267,500 FFU/mL or 267.5 FFU/ul ?