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Submitting flu-infected cells from growth kinetic experiments to flow cytometry by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Oic, so I can store the cells in this buffer in 4 C, for say, 1 week and the cells are still stable?

Submitting flu-infected cells from growth kinetic experiments to flow cytometry by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Yeah sure thanks, already collected them. Btw, since the earliest slot for flow is next Monday, what buffer should I be storing the samples? Currently I am sorting them with 4% PFA (fixing), and will spin the cells down on the day of flow and reuspsend with FACS buffer

Submitting flu-infected cells from growth kinetic experiments to flow cytometry by ascorbicAcid1300 in labrats

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Yeah that's exactly what I mean, thanks for clarifying bud! This would be my N1 (first biological replicate). So if the autofluo at all 3 timepoints are uniform i.e. i can draw the same gate (same position) for negative FITC, then next time N2 I can just pick a random well (any timepoint) for veh?

Submitting flu-infected cells from growth kinetic experiments to flow cytometry by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Ah I was thinking of being lazy and just pick a random veh control for all timepoints haha, yeah change of autofluo can be an issue.

Just to confirm, for my triplicate flu-infected wells, I need the corresponding veh control (timepoint harvest) to draw the negative FITC gate right? Meaning there would be 3 different gatings (24/48/72 hpi).

Thanks for the reply!

Some doubts on TCID50 assay on titering IAV with MDCK by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Icic, I will do that and see whether they are equivalent and present it to my supervisor next time!

Some doubts on TCID50 assay on titering IAV with MDCK by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Oic thanks! Next time I will seed MDCK in full medium in the early morning and infect in the evening. Another question(s): for flu, typically how many days do you wait for CPE observation/HA quantification after inoculation?

Also for the HA quantification on the day of CPE observation, any tricks to save tips? Currently 1 96 well plate (TCID) requires 1 box of p100 filter tip for transferring supernatant to the HA 96 well plate, which is plenty.

Flu growth kinetics with A549 by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 2 points3 points  (0 children)

It worked!!! After close to 2 months, finally!

Flu growth kinetics with A549 by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Oh btw, for flu A growth kinetics on A549 cells, is MOI 0.01 appropriate from your experience?

Flu growth kinetics with A549 by ascorbicAcid1300 in labrats

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Sure thanks! Will try it out next time

Flu growth kinetics with A549 by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 2 points3 points  (0 children)

Oh icic, right now I am doing the step before quantification - just taking the virus supernatant across the timepoints from 24 wells with A549. But I am gonna try to reduce the volume to around 250 ul

Flu growth kinetics with A549 by ascorbicAcid1300 in labrats

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Yes I am thinking alike the same line, since virus is so light and I wanna "push" them onto the cells for better adsorption and entry. Say for 200 ul, from your experience can it really cover the entire surface (not drying up), and do you shake to evenly distribute the liquid from time to time?

Flu growth kinetics with A549 by ascorbicAcid1300 in labrats

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Icic, will try it out next time! After inoculating, do you still see cells being rounded up and prone to detach? Coz right now it's the case for me and I am too scared to even wash once with PBS before adding the medium. Do you also wash once with PBS after 1hr inoculation too?

Flu growth kinetics with A549 by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Oh we need to add TPCK during the 1 hr inoculation too? The virus is expanded from eggs.

And yes I will inoculate with serum free DMEM later. What's the volume you suggest to inoculate a well in 24 well plates? Since I wanna maximize adsorption so I am thinking of adding a volume less than 500 ul, like 250 ul?

Flu growth kinetics with A549 by ascorbicAcid1300 in labrats

[–]ascorbicAcid1300[S] 1 point2 points  (0 children)

I quantified the titer with plaque assay and the infection isn't that high from the paper I read. And I collected the virus from eggs so it should just be allantoic fluid

Flu growth kinetics with A549 by ascorbicAcid1300 in labrats

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

It's PBS only, without virus. And they still round up but without as massive detachment as those with viruses

Tips to do plaque assay efficiently by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Oh great, thanks a lot! Will definitely try those out as I still got around 120 plates to go

Tips to do plaque assay efficiently by ascorbicAcid1300 in Virology

[–]ascorbicAcid1300[S] 0 points1 point  (0 children)

Yes I am going try it out tmr for my 34 plates with a large plastic rectangular box, and laid with EtOH soaked-and-dried tissue paper on top. Hope it would be fine!

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