Advice for analysing longitudinal study samples by flow cytometry

[u/Haush]

I am planning an experiment where we want to know how much a particular cell surface protein (protein X) levels change with our drug treatment. Our drug should remove this protein. We will measure blood immune cells prior to drug administration, and then at different time points over time after administration. Ideally we would have a vehicle control group for each time point, and that would be our reference. But we can't do that in this case. So we will have to compare the levels of Protein X at each time point, to the pre-dose measurements for each animal.

Here's some other design points:

• We will use the same flow cytometer for all measurements, a conventional Fortessa, and it will be properly calibrated and maintained
• We plan to have a 'biological reference' measurement at each time point; this will be cryopreserved cells that will not change over time. At each time point, we'll thaw one of these and then do a stain for Protein X using the same cocktail as other samples. This should control for variations in staining and analysis.
• We will also include FMOs for Protein X for every sample, every time point. MAybe every sample is over kill, but this will be the 'zero' or background fluorescence.

I'm keen to hear from flow cytometry experts; is this plan sound? Would you recommend anything else?

[u/skipper_smg]

I would include Sphero Rainbow Beads to monitor and standardize the instrument settings independent of CS&T. Maybe thats already included but the consensus as to what „properly calibrated and maintained“ means differs quite a lot.

[u/btags33]

If you can include mesf or abc beads instead of our in addition to the biological control so you can do some quantitation of the protein expression.

[u/Haush → u/btags33]

Thanks, yeah I was wondering if these were necessary or not. I haven't used them before but it looks pretty straight forward.