Dear all,
I realized that Biolegend has all its Treg staining kits as AF488 conjugated FoxP3 and other dyes for CD25/CD4. What is the reason behind it that they are changing CD25 and CD4 fluorochrome but not FoxP3? Is AF488 is more stable (or better) for ICFC, especially FoxP3?
I also wonder that this specific difference between FITC and AF488 because one of my friends is staining CD68-AF488. She differentiates monocytes to macrophages, then stains them w/ CD68-AF488 but the cells are 58% positive for it. On the other hand the same cells are 90% positive for CD11b-FITC. So, is AF488 not a good candidate for ICFC, or better than FoxP3? I am asking this because APC, APC/Cy7 and PE are already selected for other markers.
Thanks in advance.
Dear Oğuzhan Köse ,
I suppose it is acceptable to use anti-Foxp3-AF488 for intracellular staining of primary human T cells. AF488 is somewhat brighter and more photo-stable than FITC, meaning that you could take a longer time for analyzing FoxP3 expressing cells by using anti-Foxp3 AF488. However, if you wish to have a brighter alternative, I could recommend anti-Foxp3 APC or AF647.
On the other hand, you had better be careful about the choice of anti-Foxp3 clone. You can choose anti-Foxp3 clones, including PCH101, 259D/C7, 206D, 236A/E7, 150D and 3G3. These clones may have different characteristics, such as different staining levels, frequencies, or background staining levels. For example, you can refer to the following paper.
The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells
https://pubmed.ncbi.nlm.nih.gov/19845018/
Hi Tetsuo
As far as I know both AF488 and FITC have similar emission and excitation maxima, but the first one is more stable and brighter. In adittion, AF488 support a broader pH range, which makes more suited for intracellular staining despite its higher molecular weight compared to FITC.
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