Hi,
I would like to ask about the compensation procedure. I use Fortessa/ Diva and automated compensation procedure.
I have a set from BD for compensation setting. Referring to the manufacturer procedure, I have to mix negative beads with a positive one, stain it together, wash and run. It seems to be clear.
But I have 4 fluorochromes (antibodies) and 5th of my colour is for alive/dead cells. I use the Aqua Zombie dye, which stains the protein inside the dead cells. So it won’t work with my beads.
So the question is: During compensation can I use the beads for single staining my 4 fluorochromes, and then use cells ( dead population mixed with alive one) for my 5th tube of single staining?
What about the gating of the population? Should I create the gate of the negative population using my beads or cells?
I will be very grateful for any tips.
Hi Dominika,
Yes, you can use both beads and cells as single stains within one experiment. Set the negative gate for each single stain sample separately.
If you want to make your life even easier and don't want to always make a dead cell control, you can buy beads that are designed especially for this purpose. I use these: https://www.thermofisher.com/order/catalog/product/A10346#/A10346
Good luck!
Erinke
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