In fact, both BV421 and APC works well for CD25. Issue looks to me with the antibody titration.

I recommend going through the link printed below for you, which talks about the Stain index, for a better understanding on the importance of antibody titration for flowcytometry.

https://www.biolegend.com/ja-jp/blog/the-stain-index-what-is-it-and-what-does-it-tell-you

we did calculate a stain index for CD25 BV421, tried 1.25, 2.5, 5 and 10ul per sample. The 10ul dose had the best stain index so we've been using that amount.

First, the signals you are receiving can be background, or the fluorochrome might be brighter than the markers' expression itself.

Using APC and receiving no signal makes much more sense because usually, CD25 receptors are small in numbers, especially in Tregs. I have to ask what other markers you are detecting at the same time with CD25 because if you are, you need to consider the fluorescence spectrum overlaps.

Here are what you can do to find out about the problem. I would suggest you to use a fresh sample like peripheral blood sample instead of control, and it would be even better if you separate the buffy coat with Ficoll. This time use PE instead of APC. One of the cell types that express CD25 very brightly is the hairy cells, so you can check your fluorochrome function with that in case you have a fresh sample.

Hi everyone,

Could you please let me know if CD25 goes with PE fluorochrome?

Thanks

Why in the world it shouldn't go !! It goes definitely. However, it depends on what other conditions being used like what other fluorochromes, what type of sample, what technique etc.,

Fluorescent conjugates vary in their brightness and antigens vary in their expression levels. Highly expressed antigens will be resolved with almost any fluorophore, but antigens expressed at lower levels may require brighter fluorophores. So, always use the brightest fluorophores for low abundant proteins and vice versa to maximize sensitivity.

BW