When doing antibody titration for flow cytometry, is the unstained control sufficient as a negative control, or isotype control also required?
Good morning,
To use unstained control will be fine. Since 1998, use of isotypic controls are not recommended, but use of Fc blocking is found helpful for good results. Please see:
M. Keeney et al, Cytometry B, 1998
Morten N Andersen, Cytometry A, 2016
I also recommend you to read "https://expert.cheekyscientist.com/strengths-and-weaknesses-of-isotype-controls-in-flow-cytometry/" which is a very comprehensive summary of the situation written by Tim Bushnell.
Best wishes
Dear Emel Karakaya ,
Unstained control will work fine. Simultaneously, you can try FMO (if you want to). Either way, gating population of interest is important.
Best regards,
Babu M.
Un stained control is good. But, it is better if you could do isotope control.
As per my understanding, unstained cells are used to set voltage of the flow cytometry instrument. Unstained cells is an acceptable control for majorly cell cycle based analysis. However, it depends on the type of experiment anyone is performing. Isotype control is needed as well as fine. The only secondary antibody also uses by many if its two-step protocol.
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Fluorescence minus one controls are very useful for knowing where exactly to set your gates by accounting for spectral overlap between fluorophores, especially when it is a complex experiment that you are not sure about gating.
After that, unstained control is fine.
Hello everyone ... Thank you very much for your answers to my question. You often said that when performing antibody titration for flow cytometry, unstained control is sufficient as a negative control and no isotype control is required. I also think like you. But the friend responsible for flow cytometry and analyzing at our university also asked me to prepare isotype control. The following thing is stuck in my head: If isotype control is used, which negative median should be used when calculating the "staining index" after titration? Unstained control or isotype control?
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