Over high percentage of CD4+CD25+‏ - (Aug/05/2010 )

Hi,

I've been struggling to use FACS and need some advice.

I'm having trouble gating CD4+CD25+ Treg cells and also when I gate, the percentage of CD4+CD25+ T cells gated in CD4+ T cells is over high (>20%). The theoretical percentage should be lower than 20% in most articals.

I'm using the following antibodies for my two-color staining. Am I using the wrong negative control? Should I use PE-Cy7-labeled Rat IgG1, l Isotype Control as my nagative control? Please advice of any error.

1) Cell line: Mouse fresh splenocytes and thymocytes
2) Antibody: FITC Rat Anti-Mouse CD4 (Cat#:553729)
PE-Cy™7 Rat Anti-Mouse CD25 (Cat#:552880)
Anti-mouse cd16/32 (Fc Block)
3) Control: Non-stained Mouse fresh splenocytes and thymocytes

Thanks

-espanababy-

Hello

How are you excluding your dead cells? Can you show us some pics of your gating? FSC/SSC etc?
Clare

espanababy on Thu Aug 5 20:12:01 2010 said:

-Clare-

What isotype are your antibodies to CD4 and CD25? If they are the same isotype as each other then you'd only need one isotype control? If not then you'd possibly need an isotype control for each of your antibodies.

Have you done any blocking/fixing steps in your protocol?

-Piersgb-

Piersgb on Fri Aug 6 10:13:55 2010 said:

The isotype for my antibody to CD4 is Rat (LEW) IgG2b, κ, and the isotype for CD25 is Rat (OFA) IgG1, λ.

My experiment purpose is to analyze the percentage of CD4+CD25+ Treg cells in CD4+ T cells.

I did block the Fc receptor with Anti-mouse cd16/32 (Fc Block), but I didn't fix it because we used the fresh cells for analysis. Should I fix it?

Is there any classical FACS protocol for analyzing CD4+CD25+ Treg cells available?

Thank you very much ~

-espanababy-

Clare on Fri Aug 6 09:14:28 2010 said:

Hello

How are you excluding your dead cells? Can you show us some pics of your gating? FSC/SSC etc?
Clare

espanababy on Thu Aug 5 20:12:01 2010 said:

Hi,

I attach part of my data here. You will see the percentage of CD4+CD25+ Treg in CD4+ T cells is 44.8 (much higher than 20%).

I just dropped my sample to Flow Cytometry Core without excluding the dead cells. Is there anything I should do to erase this high back groud?

Thank you ~

-espanababy-

Hello

Thanks for posting the pics - that makes things much easier!

Firstly, I think your FSC/SSC gate is much too big. You are looking at lymphocytes which are quite tiny! Those "bigger cells" in your FSC/SSC plot could be dying cells, so will stain positive (and this could be why you are getting 44% positive). I suggest you include a dead cells stain like PI and then gate on live cells before you look at CD4 and CD25 expression.
Does that make sense? I am completely brain dead and exhausted today!

Clare

-Clare-

Clare,

Thank you so much. I will take your advice to repeat. On the other hand, should I buy the isotype antibody and use it as my nagative control?

Thank you again~

Clare on Mon Aug 9 11:51:49 2010 said:

Hello

Thanks for posting the pics - that makes things much easier!

Firstly, I think your FSC/SSC gate is much too big. You are looking at lymphocytes which are quite tiny! Those "bigger cells" in your FSC/SSC plot could be dying cells, so will stain positive (and this could be why you are getting 44% positive). I suggest you include a dead cells stain like PI and then gate on live cells before you look at CD4 and CD25 expression.
Does that make sense? I am completely brain dead and exhausted today!

Clare

-espanababy-

Just repeat the experiment and see how you go. I never used isotype antibodies for FACS as I always had a negative control sample.
Clare

espanababy on Tue Aug 10 20:00:15 2010 said:

Clare,

Thank you so much. I will take your advice to repeat. On the other hand, should I buy the isotype antibody and use it as my nagative control?

Thank you again~

Clare on Mon Aug 9 11:51:49 2010 said:

Hello

-Clare-