Jan 14, 2026
FOXP3 Intracellular Staining Procedure
This protocol is a draft, published without a DOI.
- Micah Hunter-Chang1
- 1Beirne B. Carter Center for Immunology Research
- Cytometry
Protocol Citation: Micah Hunter-Chang 2026. FOXP3 Intracellular Staining Procedure. protocols.io https://protocols.io/view/foxp3-intracellular-staining-procedure-hk5yb4y7x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 13, 2026
Last Modified: January 14, 2026
Protocol Integer ID: 238488
Keywords: staining procedure biolegend foxp3 ic stain protocol, foxp3 intracellular, staining procedure
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Biolegend FOXP3 IC Stain Protocol
Troubleshooting
Safety warnings
**Note: FOXP3 Perm Buffer (10x) may exhibit crystallization or precipitation when stored at 2-8 °C. This is normal and does not affect the buffer's performance. Heavy precipitation observed after dilution to a 1x solution can be cleared by filtration.
Before start
**Note: For flow cytometric staining, True-Nuclear™ Transcription Factor Buffer Set (Cat. No. 424401) offers improved staining and is highly recommended over the FOXP3 Fix/Perm Buffer Set (Cat. No. 421403).
Protocol
Perform cell surface staining as described in Cell Surface Flow Cytometry Staining Protocol.
Prepare a 1x working solution of FOXP3 Fix/Perm Buffer by diluting 1 part FOXP3 Fix/Perm Buffer (4x) (Cat. No. 421401) in 3 parts PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2).
Add 1 mL of 1x FOXP3 Fix/Perm solution to each sample. Vortex, then incubate at room temperature in the dark for 20 minutes. Spin at 250 x g for 5 minutes and discard the supernatant.
Add 1 mL of Cell Staining Buffer (Cat. No. 420201) to wash cells. Spin at 250 x g for 5 minutes and discard the supernatant.
Prepare a 1x working solution of FOXP3 Perm Buffer by diluting 1 part FOXP3 Perm Buffer (10x) (Cat. No. 421402) to 9 parts PBS.
Wash once with 1 mL of 1x FOXP3 Perm Buffer.
Resuspend cells in 1 mL of 1x FOXP3 Perm Buffer and incubate at room temperature in the dark for 15 minutes. Spin down cells and discard the supernatant. Then resuspend the pellet in 100 µL of 1x FOXP3 Perm Buffer.
Add an appropriate amount of fluorochrome-conjugated antibody and incubate at room temperature in the dark for 30 minutes.
Wash twice with Cell Staining Buffer, and resuspend in 0.5 mL of Cell Staining Buffer. Then analyze with a flow cytometer.