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Fluorescence Compensation


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3 replies to this topic

#1 borriello.87

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Posted 25 September 2009 - 11:55 AM

Hi all,
suppose that I want to stain Th17 lymphocytes with IFNg-FITC and IL17-PE, do I have to stain these cells with the same antibodies separately for fluorescence compensation or can I stain them (or maybe other cells like PBMCs) with different antibodies conjugated with FITC and PE (like CD45-FITC and CD45-PE)?

Thanks!

#2 miBunny

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Posted 26 September 2009 - 05:01 PM

You can use different antibodies (I like CD4 for compensation studies). You want a good bright staining marker for compensation (you need good separation between the stained and the unstained cellsO. You don't want a marker that stains all the cells (you want to be able to compare the stained population to the marker negative population when you do the compensation).

Good luck! I can't wait to hear how the staining goes! My fingers are crossed :D

#3 borriello.87

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Posted 27 September 2009 - 11:23 AM

You can use different antibodies (I like CD4 for compensation studies). You want a good bright staining marker for compensation (you need good separation between the stained and the unstained cellsO. You don't want a marker that stains all the cells (you want to be able to compare the stained population to the marker negative population when you do the compensation).

Good luck! I can't wait to hear how the staining goes! My fingers are crossed :D


Dear miBunny,
thank you for your answer!
So....if I want to stain Th17 with IFNg-FITC and IL17-PE, is it possible to use this protocol?

1)Th17 + isotype matched antibody-FITC + isotype matched antibody-PE
2)PBMCs + CD4-FITC
3)PBMCs + CD4-PE
4)Th17 + IFNg-FITC + IL17-PE

Maybe my questions are not so clever :D , but these are my first flow cytometry experiments and I want to remove
as many doubts as possible....so thank you for your help!

#4 Binchen

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Posted 30 September 2009 - 06:33 PM

You'll also need unstained cells to set your voltages. For compensation you either need single stained controls or (much easier to gate on) compensation beads. For example anti-rat beads if your antibodies are from rat. The beads will bind the constant part of the antibody and give nice, separated positive and negative populations, which makes compensation much easier!
For gating it is good to have fluorescence minus one (all colours except the one you want to gate on) controls. They give you the combined background of all your other colours, so that you can confidently place your gate for your positive population then.

Hope that helps and doesn't confuse you even more!




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