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Materials Provided:
1. Alexa Fluor® 488 anti-human FOXP3
25 tests
2. Alexa Fluor® 488 Mouse IgG1, k isotype control
25 tests
3. FOXP3 Fix/Perm buffer and FOXP3 Perm buffer
100 tests
4. anti-human CD4 PE-Cy5/CD25 PE Cocktail
50 tests
Materials not included:
Cell Staining Buffer (Cat. No. 420201)
Immunofluorescence Staining Procedures:
Centrifugation steps: perform at 250Xg for 5min
Incubation steps: perform at room temperature.
1. Distribute 0.5-1 X 106 cells/100 μl/tube into FACS tubes.
2. Add 20 μl of CD4 PE-Cy5/CD25 PE cocktail to each tube, vortex and incubate in the dark for 20 minutes.
3. Wash once with cell staining buffer (Cat. No. 420201); centrifuge, then remove the supernatant.
4. Prepare 1X buffer solutions:The FOXP3 Fix/Perm buffer (4X) must be freshly diluted by diluting one (1) part FOPX3 Fix/Perm buffer (4X) with three (3) parts PBS. The FOXP3 Perm buffer (10X) should be diluted by diluting one (1) part FOXP3 Perm buffer (10X) with nine (9) parts of PBS.
NOTE: The FOXP3 Perm buffer (10X) may have crystalization or precipitation observed when it is stored at 2-8°C, however, it is normal and does not affect the buffer performance. If there is heavy precipitation observed after diluted to 1X working solution, it can be filtered to clarify the solution.
Caution: The FOXP3 Fix/Perm buffer contains paraformaldehyde, which is toxic and mutagenic. Please wear necessary protection to avoid direct body contact.
5. Add 1 ml of 1X BioLegend's FOXP3 Fix/Perm solution to each tube, vortex and incubate in the dark for 20 minutes, then centrifuge and remove the supernatant. The cell pellet will now be translucent and difficult to see; take care not to dislodge and accidentally aspirate cells at all later stages of staining protocol.
6. Wash: resuspend cells in cell staining buffer (Cat. No. 420201); centrifuge, then discard the supernatant.
7. Wash: resuspend in 1ml 1X BioLegend's FOXP3 Perm buffer; centrifuge, then discard the supernatant.
8. Resuspend cells in 1ml 1X BioLegend's FOXP3 Perm buffer, incubate in the dark for 15 minutes; centrifuge, then discard the supernatant. Resuspend the pellet in 100 µl of 1X BioLegend's FOXP3 Perm buffer.
9. Add 5 μl Alexa Fluor® 488 FOXP3 antibody or 5 μl Alexa Fluor® 488 mouse IgG1, k isotype control into appropriate tube and incubate in the dark for 30 minutes.
10. Wash twice with cell staining buffer (see step 6) then resuspend in 0.5ml cell staining buffer . Analyze with flow cytometer using appropriate instrument settings.
* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm.
** Alexa Fluor® is a registered trademark of Molecular Probes, Inc. Alexa Fluor® dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents. |
