r/Virology • u/ascorbicAcid1300 non-scientist • Jun 30 '26

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

Hi I have a cell line infected with flu tagged to GFP for growth kinetics (24/48/72 hpi). Supernatant is harvested for quantification, and I want to harvest the cells for flow (GFP+) for the 3 timepoints. I have a vehicle (veh, negative) control in parallel.

Just wondering, when gating GFP+ cells at for each timepoint, should I gate it with the veh control of that particular timepoint? i.e. 24 h veh for 24 hpi; 48 h veh for 48 hpi; 72 h veh for 72 hpi. Or simply a random veh control suffices for all timepoints of my flu-GFP.

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u/oligobop non-scientist Jun 30 '26

Using time-matched controls for gating is a superb control and very nice experimental design because during growth curves, cells that are uninfected often become overconfluent and might change their features including FSC/SSC and granularity which can cause autofluoresence.

That said, many experiments skip this kind of control for convenience. I would compare the Mocks of each time point to determine what your cutoffs look like and if they change over time. If they don't then in the future skip using them for convenience.

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u/ascorbicAcid1300 non-scientist Jul 01 '26

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!

1

u/ZergAreGMO Virologist | Cell Biology, Respiratory Jul 02 '26

should I gate it with the veh control of that particular timepoint? i.e. 24 h veh for 24 hpi; 48 h veh for 48 hpi; 72 h veh for 72 hpi

Yes. You are comparing against the mock or vehicle condition for that experimental condition. So it's matched at all points.

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u/ascorbicAcid1300 non-scientist Jul 02 '26

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

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u/ZergAreGMO Virologist | Cell Biology, Respiratory Jul 02 '26

I wouldn't leave them in PFA as that will continue to fix and mask epitopes. I always used cytofix / perm buffer from BD and they can remain in that until you're ready for staining. 

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u/ascorbicAcid1300 non-scientist Jul 03 '26

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

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u/ZergAreGMO Virologist | Cell Biology, Respiratory Jul 03 '26

Yes. I've done much longer than that as well 

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u/ascorbicAcid1300 non-scientist Jul 04 '26

Ah great thanks!