3.1 Nuclear Factor Staining – Taking Regulatory T Detection as an Example
Surface Staining
Prepare a single-cell suspension (1×10⁶ cells) in 100 μl of Staining Buffer. Stain with CD4-FITC and CD25-PE antibodies, then incubate at 2-8°C in the dark for 20-30 minutes. Set up blank tube, compensation tube, control tube, and sample tube.
Fixation/Permeabilization
Vortex to mix the cells thoroughly, add 1 ml of freshly prepared 1X Fix/Perm Working Solution, mix well, and incubate at 2-8°C in the dark for 40-50 minutes.
Permeabilization/Washing
Add 1X Perm/Wash Buffer to the fixed and permeabilized cells, centrifuge at 350×g for 6 minutes at 2-8°C, and discard the supernatant. Add 2 ml of 1X Perm/Wash Buffer again, centrifuge at 350×g for 6 minutes at 2-8°C, discard the supernatant, and repeat this washing step once more (add 2 ml of 1X Perm/Wash Buffer, centrifuge at 350×g for 6 minutes at 2-8°C, then discard the supernatant).
Intranuclear Staining
Resuspend the cells in 80-100 μl of 1X Perm/Wash Buffer, add FOXP3-APC-conjugated antibody or non-specific control, vortex for 10 seconds to mix thoroughly, and incubate at 2-8°C in the dark for 40-50 minutes.
Permeabilization/Washing
Gently vortex to mix the samples before washing, add 2 ml of 1X Perm/Wash Buffer, centrifuge at 350×g for 6 minutes at 2-8°C, and discard the supernatant.
2. Case of Human Peripheral Blood Treg Staining:
Flow cytometric analysis: Human PBMC were co-stained with FITC Anti-Human CD4 and PE Anti-Human CD25. The cells were fixed and permeabilized, followed by intracellular staining with Alexa Fluor®-conjugated antibodies. Based on the light scattering characteristics of lymphocytes and the fluorescence characteristics of CD4⁺ or CD25⁺ cells, the results are presented as dot plots of FoxP3 vs. CD25 (left panel) or FoxP3 vs. CD4 (right panel). Flow cytometry was performed on a BD FACSCalibur™ System.
