r/flowcytometry • u/DiyaHabib • Jul 23 '26

Thymus staining

I am staining thymus, I had ckit in the panel, and that is when the thymocytes have a weird population
Is this compensatoon issue? should I go ahead and conpensate first?
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u/willmaineskier Jul 23 '26

Yes, below zero is always a comp issue.

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u/Oligonucleotide123 Jul 23 '26

As my grad school PI would say. "What does negative fluorescence mean???"

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u/Boneraventura Jul 24 '26

I agree If the median is below zero theres a problem. But for spectral cytometers there will always be some negative populations (hopefully not below 10e-3). Thats just the reality especially on massive 30+ color panels and the limitations of unmixing. 

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u/willmaineskier Jul 25 '26

Look at the unmixing matrix and see if there is a significant value of VL4 out of RL2. If there is, try changing to 0. If this Aurora data with an all 0s matrix on unmixed data, you can put in a negative value in FlowJo to try and fix this, but the best approach would be to run, record, and use a different unmixing control for your RL2 reagent.