Foxp3 / Transcription Factor Staining Buffer Kit

Applications:

FC

Cat no : PF00011

---

Validation Data Gallery

• 
• 

  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).

• 

  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).

---

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 Ki-67.

This product contains formaldehyde, FBS, and sodium azide (≤ 0.09%).

Components

Components	Size
Foxp3 / Transcription Factor Fix/Perm Concentrate (4X)   PF00011-A	30 mL
Foxp3 / Transcription Factor Fix/Perm Diluent (1X)   PF00011-B	100 mL
Flow Cytometry Perm Buffer (10X)   PF00011-C	100 mL

Storage

Store at 4°C for up to 6 months.

Protocol

Prepare working solutions:

Fix/Perm Buffer:	FC Perm Buffer:
1 mL needed for each sample of 1x10^6 cells; freshly prepared 1 part Transcription Factor Fixation/Permeabilization Concentrate (4X) (PF00011-A) 3 parts Transcription Factor Fixation/Permeabilization Diluent (PF00011-B)	4-5 mL needed for each sample Dilute Flow Cytometry Perm Buffer Concentrate (10X) (PF00011-C) to 1X with distilled water

Additional Reagents Recommended:

Flow Cytometry Staining Buffer (PF00012)

Protocol:

1. Prepare working solutions as described in the table above.

2. Stain any cell surface markers in the panel using the Proteintech Flow Cytometry Cell Surface Staining Protocol. (If panel does not include cell surface markers, skip this step.)

3. (After staining cell surface markers, wash and remove the supernatant.) For each tube of 1 x 10^6 cells, add 1 mL Fix/Perm Buffer. Gently pipette to fully suspend cells.

4. Incubate for 45 min at RT in the dark.

5. Centrifuge at 400-600 g for 5 minutes. Aspirate the supernatant.

6. Add 2 mL FC Perm Buffer. Gently pipette to fully resuspend cells.

7. Centrifuge at 400-600 g for 5 minutes. Aspirate the supernatant.

8. Resuspend cell pellet in 100 uL FC Perm Buffer and incubate for 10-15 min at RT in the dark.

9. Using the vendor recommended amount of antibody, for example 5 uL/test, (or pre-dilute flow cytometry antibodies with FC Perm Buffer to the optimal titration), incubate the cells with the antibodies for 30-45 min at RT in the dark to stain intracellular targets.

10. Wash cells by adding 2 mL FC Perm Buffer. Centrifuge at 400-600 g for 5 minutes. Aspirate the supernatant.

11. Repeat step 10.

12. Resuspend cell pellet in 0.2 mL FC Perm Buffer.

13. Analyze cells with a flow cytometer.

Notes

1. This product contains formaldehyde and sodium azide. Take protective measures and avoid contact with eyes and skin.

2. During incubation, make sure cells are mixed well and prevent cells from sticking to the side of the tube to prevent false negative populations.

3. Take care to avoid light during this protocol.

Cited in Article as

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