Hi,
Could someone please tell me what it means when there are 'very negative' populations in multi-colour (8+) flow cytometry?
Example:
- I have attached a plot, with Cy5 on the x-axis.
- I compensated using compensation beads, and updated the matrix as necessary.
Is it simply a compensation issue or is it something else?
Thank you,
Did you try to change the voltage of cy5 laser? By changing it you should be able to make your supernegative population higher than zero
Overcompensation! Do you have a granulocyte marker in another fluorochrome? Do you want to check your compensation values in the real world of your fully stained sample? There's a good story that tells: always put in a bivariate plot all stains one vs another and let them talk!
Hi Marco, Thank you.
I didn't change the Cy5 voltage as these are automatically adjusted using CS&T beads for consistency between experiments.
Thanks Mario.
No, I don't use a granulocyte marker.
The Cy5 signal is coming from nanoparticles which are likely associating with granulocytes (plus monocytes, and so on).
the NP are mostly in monocytes based of the plot- what are the characteristics of the NP's?
Hi, just saw your question. Overcompensation is not the only way to get this result. It could be a baseline restore issue. I presume this was run using DiVA? Have a look at the uncompensated Cy5. Does the negativity go away? If so, it's a comp issue. If not, you may have created an artefact on the machine. This wouldn't be a big surprise since you're using nanoparticles. I used to use qdots to reproduce the effect when we were looking at it a few years ago. Also recommend that you look at Cy5 vs. time. It's possible it's just some end-of-run weirdness from running the sample dry.
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