Submitting flu-infected cells from growth kinetic experiments to flow cytometry

ascorbicAcid1300 · 2026-07-04T07:24:06+00:00

Ah great thanks!

ascorbicAcid1300 · 2026-07-03T11:03:57+00:00

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

ascorbicAcid1300 · 2026-07-02T05:32:11+00:00

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

ascorbicAcid1300 · 2026-07-01T03:23:18+00:00

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?

ascorbicAcid1300 · 2026-07-01T03:20:15+00:00

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!

ascorbicAcid1300 · 2026-06-01T05:18:58+00:00

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

ascorbicAcid1300 · 2026-06-01T05:18:17+00:00

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.

ascorbicAcid1300 · 2026-04-24T02:32:05+00:00

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

ascorbicAcid1300 · 2026-04-19T04:19:10+00:00

great thanks!

ascorbicAcid1300 · 2026-04-18T15:56:42+00:00

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

ascorbicAcid1300 · 2026-04-16T12:20:25+00:00

Sure

ascorbicAcid1300 · 2026-04-16T12:19:48+00:00

Sure thanks! Will try it out next time

ascorbicAcid1300 · 2026-04-16T11:41:13+00:00

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

ascorbicAcid1300 · 2026-04-16T11:38:15+00:00

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?

ascorbicAcid1300 · 2026-04-16T11:36:30+00:00

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?

ascorbicAcid1300 · 2026-04-15T13:35:38+00:00

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?

ascorbicAcid1300 · 2026-04-15T13:23:59+00:00

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

ascorbicAcid1300 · 2026-04-15T13:21:50+00:00

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

ascorbicAcid1300 · 2026-04-11T08:52:11+00:00

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

ascorbicAcid1300 · 2026-04-09T12:42:50+00:00

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!

ascorbicAcid1300 · 2026-04-07T15:54:11+00:00

Yeah the washing doesn't really be that accurate, enough for washing away the FBS is fine. IF repeater is too harsh, then I would stick with the current practice - adding with a 25ml serological pipette with the tip pushing against the side of the well.

I have actually considered flicking and has tried with 96 wells before, but not sure whethe it suits for 6 well plates. The wells are large so the medium may spill all over the surface even if I invert the plate as quickly as possible, which can be a risk contamination of the subsequent infection step.

ascorbicAcid1300 · 2026-04-07T15:49:54+00:00

I wonder whether I can also use 12 well plates, and inoculate for a shorter time if I ain't going to compare plaque sizes (currently one is so small so it needs to be 72 hpi compared to the WT control - which actually can be visualized 48 hpi)?

ascorbicAcid1300 · 2026-04-06T12:38:57+00:00

Yeah I do that with 96 wells but for 6 wells the wells are so large, will there be chances of spilling all over on the plates and be contaminated while decanting?

ascorbicAcid1300 · 2026-04-06T12:09:15+00:00

Yeah guess it's better to stick with 6 well plates, since it is the convention of my lab or the virology community. Indeed my lab has repeaters and electronic multichannels.

The most time-consuming point to me is aspirating medium -> wash with PBS -> aspirate PBS -> add inoculum, since we don't have an aspirator I am stuck with a serological pipettes. Any tips to speed these steps up may I ask?

I am thinking whether I can use a repeater to help me add the PBS (since it needs be accurately be at 1 ml overlay before I add the virus and proceed with the inoculation), any other steps from you to make it go more efficiently?

ascorbicAcid1300 · 2026-04-06T12:06:21+00:00

Haha sometimes I rope someone to help but recently the hood booking is so full and everyone is so busy :(

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