Foxp3 / Transcription Factor Staining Buffer Kit

Applications:

FC

Cat no : PF00011

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Validation Data Gallery

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  1x10^6 Mouse splenocytes were intracellularly stained with 0.06 ug APC Anti-Mouse Foxp3 (APC-65089, Clone:3G3), and 0.06 ug CoraLite® Plus 488 Anti-Mouse CD4 (GK1.5) (CL488-65104, Clone: GK1.5), and 0.06 ug APC Mouse IgG1 Isotype Control (MOPC-21) (APC-65124, Clone: MOPC-21). Cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Kit(PF00011).

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  1x10^6 Human PBMCs were intracellularly stained with 5 ul APC Anti-human Foxp3, and 5 ul FcZero-rAb™ CoraLite® Plus 488 Anti-Human CD4 (CL488-FcA98042, Clone: 240427E12), and APC Mouse IgG1 Isotype Control (MOPC-21) (APC-65124, Clone: MOPC-21). Cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Kit(PF00011).

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Product Information

The Foxp3 / Transcription Factor Staining Buffer Kit contains specially formulated buffers and solutions for optimal resolution and low background in your analysis of nuclear antigens by flow cytometry. Included are the following components, to be used during staining protocols for detection of nuclear antigens such as Foxp3 and ROR gamma.

Components

Components	Size
Foxp3 / Transcription Factor Fix/Perm Concentrate (4X)   PF00011-A	50 mL
Foxp3 / Transcription Factor Fix/Perm Diluent (1X)   PF00011-B	160 mL
Flow Cytometry Perm Buffer (10X)   PF00011-C	150 mL

Storage

4°C

Protocol

1. Prepare working solutions:

1) Working Solution D: Take 1 part of PF00011-A (Foxp3 / Transcription Factor Fix/Perm Concentrate 4X) and 3 parts of PF00011-B (Foxp3 / Transcription Factor Fix/Perm Diluent 1X ) , and mix them.

(2) Working Solution F: Take 1 part of PF00011-C (Flow Cytometry Perm Buffer 10X) and 9 parts of distilled water and mix well to prepare Flow Cytometry Perm Buffer (1X).

2. Perform cell surface staining as described in the Proteintech Cell Surface Flow Cytometry Procedure.

3. After a final wash, discard the supernatant and add 2 mL of Working Solution D to each EP tube, blowing slowly to ensure complete resuspension of the cells. Incubate for 45 minutes at room temperature away from light.

4. Centrifuge at 400-600 g for 5 minutes at room temperature, discard supernatant, add 2 mL of Working Solution F to the EP tubes, and blow slowly to ensure complete resuspension of cells.

5. Resuspend the cell precipitate in 100 μL of Working Solution F and stand for 10-15 minutes.

6. Using the recommended amount of antibody (or pre-dilution of flow cytometry antibodies using Working Solution F) to incubate the antibody-cell mixture for 30-45 minutes away from light, for detection of intracellular antigen. 

The recommended amount of antibody: e.g., 5 μL/Test.

7. After incubation, add 2 mL of Working Solution F to each tube.

8. Centrifuge the tubes at 400-600 g for 5 minutes at room temperature and discard the supernatant.

9. Add 2 mL of Working Solution F to each tube.

10. Centrifuge tubes at 400-600 g for 5 minutes at room temperature and discard supernatant.

11. Resuspend the stained cells in 0.2 mL of Working Solution F.

12. Collect samples on a flow cytometer.

Expiration Date

12 months after receiving.

Cited in Article as

PF00011, Foxp3 / Transcription Factor Staining Buffer Kit, Proteintech, IL, USA