Strange serum signal - plateK12rep1_20200430_155932_313 - H01-0004

[tischi]

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@metavibor @imagirom

I am looking at one of the well where you did not "add the serum". Could be explain again, what you added excatly?

1. I guess you still added the secondary antibody with the fluorophore, right?
2. The pattern I observe does not look like channel crosstalk at all!
3. It rather looks as if the secondary antibody binds to infected cells.

Screenshot

[metavibor]

@tischi @imagirom hmm that could explain the slightly above 1 ratio we always have... yes I added secondary antibodies but starting from plate K17 I stopped doing this, now I have "no serum, no secondaries" in H1... We now have a number of H1 wells with or without secondaries, perhaps it is worth doing analysis of background in infected and control cells,.. just to see how much different is it... if significantly different we could subtract different background values from different cells and get the ratio precisely to 1 for negative controls

[tischi]

if significantly different we could subtract different background values from different cells and get the ratio precisely to 1 for negative controls

Yes, that would be in fact another (maybe even more accurate way of background correction). I am just rephrasing: Measure the mean intensity in H1 inside infected and non-infected cells and subtract this from the respective populations. I like it! We should definitely try it!

[tischi]

@metavibor Is that something potentially interesting to follow up biologically at some point? It seems as if the secondary antibodies (hopefully not the fluorophores) seem to recognise something in the infected cells...