88-5778

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Contents: hFOXY Staining Buffers (perm/fix/block)
Catalog Number: 88-5778
Storage Conditions: Store at 4°C.

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Description

Please see the following link for FAQ regarding the usage of eBioscience Foxp3 reagents.

http://www.ebioscience.com/ebioscience/Foxp3FAQs.htm

The eBioscience hFOXY Staining Buffer Set has been formulated and optimized for staining isolated human PBMC with hFOXY monoclonal anti-human Foxp3.
The set components Include:

1-Solution A: 100mls
2-Solution B: 100mls
3-eBioscience Flow Cytometry Staining Buffer: 200mls
4-Blocking Buffer: 4mls

Intracellular Staining Protocol for hFOXY (anti-human Foxp3) on isolated Human PBMC

1. Isolate PBMC from human peripheral blood.
2. Stain surface molecules such as CD4, CD8, etc. using the surface staining protocol: http://www.ebioscience.com/ebioscience/appls/FCS.htm. Wash with staining buffer, spin and remove supernatant.

Note: We have found that for some cell-surface antigens including CD4 and CD8 (using clones RPA-T4 and RPA-T8, respectively) surface staining can be performed simultaneously with hFOXY after treatment with Solutions A and B. Furthermore, some antigens, including CD25, are altered during the staining protocol resulting in decreased binding of antibody. Some clones are more susceptible to these alterations and individual monoclonal antibodies must be tested to determine the most appropriate time for staining.

3. Add 1ml of Solution A per approximately 10⁷ cells while vortexing the tube. Incubate in the dark at room temperature for 20 minutes.
4. Incubate the cells at -20 degrees C overnight.
5. The next day, thaw cells and centrifuge to remove Solution A.
6. Add 1ml chilled Solution B per approximately 10⁷ cells. Incubate in the dark at 4 degrees C for 20 minutes.
7. Centrifuge cells and completely remove Solution B by aspiration.
8. Wash cells in eBioscience Flow Cytometry Staining Buffer. Spin and decant supernatant.
9. Repeat step 8.
10. Resuspend 10⁷ cells in 1ml of Flow Cytometry Staining Buffer.
11. Dispense 10⁶ cells (~100µl) per test tube.
12. Add 20µl of Blocking Buffer to each tube; incubate in the dark at room temp for 15 minutes.
13. Stain with conjugated anti-human Foxp3 antibody. Add 20µl of pre-titrated antibody to the appropriate tube. Please perform further titration for optimal staining in your own assay system.

14. Incubate 20-30 minutes in the dark at room temperature.
15. Add 1 ml of Flow Cytometry Staining Buffer to each tube, centrifuge for 5 minutes at 400x g and aspirate supernatant.
16. Repeat step 15.
17. Resuspend the cell pellet in 0.2 ml of Flow Cytometry Staining Buffer. Analyze on cytometer.

Applications Reported

For research use only, not for diagnostic or therapeutic use. hFOXY Staining Buffers has been used in intracellular flow cytometric analysis with hFOXY mAb.