Hello Fellow Scientists,
We want to analyze regulatory T cells (CD3/CD4/CD25/FOXP3) in patients PBMCs by flow cytometry.
So far we have been unable to get any specific CD25 stain. CD3, CD4, and (after permeabilization) FOXP3 antibodies work fine.
Antibody: anti-CD25-PE and anti-CD25-PerCP/Cy5.5 from BioLegend - no specific signal above isotope control
Cells: Isolated and cryopreserved PBMCs, thaw, treated with DNAse to remove dead cells and debris, washed, rested for 15 minutes and resuspended in FACS buffer we are using (0.5% BSA, 0.1% sodium azide in PBS). Stained with different concentrations of anti-CD25 antibody in FACS buffer for 30min at 4 degrees in the dark. Analyzed on FACS Calibur or AriaII.
We also tried nonspecific stimulation (PMA/ionomycin) and no success.
Is it the antibody or we are missing something here?
I would be very grateful for your suggestions.
Best wishes,
Malgorzata
hi
Im not a Fellow Scientist , But I think it's best to do the following:
1- check CD25 antibody efficacy by positive cell line such as "DEL" cell line malignant histiocytosis ( CD25 99% +)
2- you can use from BD Pharmingen Human Regulatory T Cell Cocktail kit (cat no: 560249)
good luck
Dear Malgorzata
Did you test the anti CD25 markers with fresh PBMCs without previous treatment with DNAse?
Perhaps cryopreservation affected the expresion of CD25 and a previous in vitro culture for cells recovery could be neccesary. See the following paper (here the expresion of other markers in Tregs were affected by criopreservation of PBMCs and it was solved with in vitro culture by 4h).
Best regards
Dear Malgorzata Garstka,
It is recommended that antibody testing be first carried out with fresh ficoll isolated PBMC from healthy volunteers, without extra manipulation as suggested by Dr Alexander Batista Duharte.
A positive control is sometimes more important than negative controls (f.g. isotype controls). Your staining results with anti-CD25 PE might be compared with the staining results with anti-CD4 (or CD3, CD8, CD20, ...) PE. If both show bad results, the problem may be independent from the anti-CD25 antibody (f.g. laser, detector, filter, compensation, etc). If the former does not work while the latter works, then the batch of anti-CD25 may be a problem.
If a batch of antibody is not working, it might be necessary to test different batch or sometimes even another clone of anti-CD25. For example, BD: anti-human CD25 clone M-A251, BioLegend: clone BC96, while Miltenyi: clone 4E3. I could recommend to test the Miltenyi's 4E3 (PE is more desirable than FITC or APC) and compare the results with BC96.
Tetsuo Tsukamoto Thank you for the detailed answer.
@Alexander Batista Duharte, @Reza Hadavi thanks for your answers.
We used CD25 antibody from Biolegend - clone BC96, also with PE.
Anti-C3, anti-CD4, L243 (anti-DR) and many other antibodies using the same conditions worked. FOXP3 (after permeabilization of cells) worked.
In summary, this week we will give the last try on fresh PBMCs. Currently trying to borrow some other CD25 antibody from scientists at my university.
Otherwise we will buy Miltenyi's 4E3.
I use anti-CD25 PE from BD Biosciences and it work fine. However I feel that you will detect the CD25 in fresh cells. In addition, I suggest to read the paper that I shared with you. The authors improve the markers detection in cryopreserved cells by in vitro culture for funtion cells recovery before the FACS analysis.
Good luck!
Alexander Batista Duharte
Thank you for your answers. We have samples from patients already collected and preserved. I have read some papers about how to improve FACS stain on thaw cells - that is why DNAse treatment - to eliminate lysed cells. I will check paper you shared for more details.
Would you recommend other marker for regulatory T cells, instead of CD25? We have CD3/CD4/FoxP3 that work in our set up.
CD 127low is also used for human Tregs identification but not in sustitution of CD25.
Often is used CD4÷CD25÷CD127low as a panel for human Tregs phenotipagem. BD bioscience and Miltenyi have a kit.
Hi, as Alexander Batista Duharte has suggested the addition of CD127 to your CD3+ cells may help (CD3+, CD127 low , CD25+). Then maybe the use of CD25 from Beckman coulter may help? I use it with APC as fluorophore and get good reproducible results.
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