hello everybody,I have a question about FACs assay.
I am working on cell-cell interaction, I have some modifies cells which can interact together in presence of co factor, but the problem is that the co factor itself make the peak kinda shifted!?
foe example I have a two cell population and stained them with different dye(DiO and DiI) on FACs results we should see three population the green one the red one and the mix population (yellow) but after the co factor every peak shifted ( for a blank or control sample), then I can not say my cells interacted or not?
I will be thankful if anybody has or had experience before share with me.
thank you.
What do you mean when you added the co-factor alone? Can you give more details on your controls and test samples?
Hi Samaneh. The shift in fluorescence can be attributed to changes in background fluorescence but these shifts are not that dramatic. If two cells are interacting, despite shift in autofluorescence after addition of cofactor (if that is a nonfluorescent molecule by nature), you will see double positive cells on the top right corner of dot plot. Did you see double positive cells after cofactor? And secondly, to avoid the issue, you can take unstained cells with cofactor as control rather than unstained cells. It will be a better blank/control for your flow cytometric data.
@Ravi Kumar Sharma. hi thank you so much for your answer.yes I will see double positive cells but they are really close to each other I will try your suggestion. I hope it work.
@Tamara Lekishvili
yes I washed them twice with PBS before reaction.
@ Julie Joseph
since I stained cells, with two different color to see if these two population interact or not , but this interaction also need a co factor, when I add the co factor which is PCB , there is two pick on the channel which I expect to see cell interactions, but this co factor interfere the results.
thank you so much to both of you.
Have you incorporated a live / dead dye? If you are getting shift in every channel it may be due to stress of cells from treatment, and dying/dead cells can cause auto fluorescence in all channels. I typically use eFlour dyes from ebioscience*, they are fixable and work very well. I think it would clean up your results a bit.
*https://www.thermofisher.com/order/catalog/product/65-0865-14
Yes, it is very common problem while segregating two populations of interest.So it is better to go with single colour compensation beads to see individual positive and negative population of each fluorescent dye which had been tagged with respective population.Thus each tagged fluorescent dye will suggest it's proper position.Then we will have a clear idea of both the the population.
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