Hello. Need help with (eBioscience) FoxP3 kit. I heard the external antibodies (CD4 and CD25) can be added, AFTER the fix/perm, after overnight incubation at 4C, CONCURRENTLY with the Foxp3 Ab.
Anybody tried it?
What would be my best controls? Single stains for each? CD4+CD25 either with or without Foxp3 (All-But-One control)? Isotype controls?
What percentage to expect, in human PBMCs ?
There are many resources, where you can inform quite well regarding different surface stains using different protocols and different clones (e.g. CD4); I personally like cytobank a lot:
https://www.cytobank.org/facselect/
http://reports.cytobank.org/5/v1
Or ebioscience to have a quick look...
http://www.ebioscience.com/resources/application/flow-cytometry/clone-performance-after-fix-perm.htm
Or BD...
http://www.bdbiosciences.com/documents/antibodies_human_cellsurface_marker.pdf
Good luck and cheers
Thanks.
0.5-4% of CD4+ cells? CD3+?
I actually tried the CD4 and CD25 with the Foxp3 itself!! it seems to work fine....
Hello
The saining Protocol of human PBMCs :
Wash human PBMCs (about 1 x 106 cells per sample) with 2 mL of Flow Cytometry Staining Buffer or other BSA-containing buffer, by spinning at 300 x g for 5 minutes, using 5 mL flow cytometry tubes. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 10 minutes at 4 °C. Surface stain with CD4 and/or CD25 antibodies for 30 minutes at 4 °C. Wash cells 2x with cold 1x PBS. Resuspend cells in freshly made 1x Fixation Buffer using 0.5 mL/tube. Incubate at 4 °C for 30 minutes. Wash 2x with freshly made, cold 1x Permeabilization/Wash Buffer. Add FoxP3 antibody (or intracellular cytokines) to cells and incubate for 30 minutes at 4 °C. Wash cells 1x with cold 1x Permeabilization/Wash Buffer. Resuspend cells in Flow Cytometry Staining Buffer and analyze on a flow cytometer.
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2862733/
http://www.ebioscience.com/resources/faq/foxp3-faq.htm
https://resources.rndsystems.com/images/site/ps_04.14_foxp3-rabbit-antibody-technology.pdf
Thanks. This protocol I know.
The question is if it can also be performed ALL the stains together (both external and internal) TOGETHER at the time of the I.C.S....
Found the answer at eBioscience's site:
Q: At what stage in the protocol do you surface stain for proteins such as CD25 and CD4?
Like traditional intracellular staining protocols, we have tested surface staining before fixation. It is possible that many of these antibodies will also work post fixation but this should be tried by the researcher for individual antibody clones. For example CD4 (RPA-T4) can be stained either at the beginning of the protocol or concurrently with PCH101 (Foxp3).
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