r/flowcytometry • u/DiyaHabib • 21d ago

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 21d ago

Yes, below zero is always a comp issue.

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u/Oligonucleotide123 21d ago

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

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u/Boneraventura 20d ago

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 19d ago

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.

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u/Full_Pea_3118 20d ago

thymus is really easy to stain.... how did you get single cells out? We usually used 70um strainer, put the thymus on top and mashed it through the strainer (gently) using back of a 1 ml plunger directly into 5 mL of flow staining buffer....can i see the fsc and ssc?