2.1 Intracellular Factor Staining

Cell Surface Antigens: Such as CD3, CD4, CD8, etc.
Cytoplasmic Antigens (Intracellular Antigens): Including IL-4, IFN-γ, IL-17, Granzyme, Perforin, etc.
Nuclear Antigens (Intracellular Antigens): Including transcription-related proteins like Foxp3, RORγ, Oct3/4

1. Stimulating Cells
There are many methods for stimulating the production of cytokines in vitro. Polyclonal antibody activators are particularly important in inducing cytokine production. Such activators mainly include the following substances: phorbol ester plus calcium ionophore or ionomycin, phytohemagglutinin, staphylococcal enterotoxin B, and monoclonal antibodies directly targeting the TCR/CD3 complex (with or without antibodies targeting co-stimulatory receptors such as CD28).
Note: It has been reported that stimulation with PMA alone can lead to a decrease in the expression of CD4 on the surface of mouse T cells. Co-stimulation with PMA and calcium ionophore results in a more significant decrease in CD4 expression, and also causes a reduction in mouse CD8 thymocytes as well as mouse and human peripheral blood T lymphocytes.

2. Sample Processing: Multicolor Staining of Cell Surface and Intracellular Factors
1) Cell Culture
Prepare viable cell populations by stimulating tissue samples in vivo or cultured cells in vitro with protein transport inhibitors. After cell isolation, resuspend them in staining medium, count, and transfer to plastic tubes or microplates for immunofluorescent staining. Cells should be protected from light during staining and storage.
2). Blocking Fc receptors: Reagents that block Fc receptors can reduce non-specific immunofluorescent staining.
a. In mice, use the purified 2.4G2 antibody specific for the FcγII/III receptor to block non-specific staining caused by the Fc receptor of fluorescent antibodies. 10^6 cells were resuspended in 100μl Staining Buffer, 1 μg  FcBlock was added, and the mixture was incubated at 4°C for 15 minutes. Then the cells were washed, and specific fluorescent antibodies against cell surface antigens (properly diluted with stain buffer in advance) were added.

# 3. Cell Surface Staining  
a. Resuspend 10⁶ cells in 50 μl of Staining Buffer, add an appropriate amount of specific monoclonal fluorescent antibodies (e.g., CD3, CD4, CD14, CD19, etc.), and incubate at 4°C for 30 minutes.  
b. Add an appropriate amount of Staining Buffer to wash the cells twice (250 μl per wash for microplates, 1 ml per wash for test tubes), then centrifuge the cells at 250×g.  
c. For human cells, pre-incubate the cells with human serum or an excess of irrelevant purified immunoglobulins (Ig) from the same species in advance, and add isotype controls to block Fc receptors.  

# 4. Cell Fixation and Permeabilization  
a. Fully resuspend the cells, add an appropriate amount of Fixation/Permeabilization Solution (100 μl per well for microplates, 250 μl per tube for test tubes), and incubate at 4°C for 20 minutes.  
Note: Vortex and mix well before adding the Fixation/Permeabilization Solution to prevent cell aggregation.  
b. Wash the cells twice with 1× Wash Buffer (250 μl per wash for microplates, 1 ml per wash for test tubes).  

# 5. (Optional) Cell Fixation, Permeabilization, and Post-Fixation Storage for Subsequent Staining  
## a. Cell Fixation and Storage  
1.) Resuspend the cells in 100 μl of 4% paraformaldehyde (or 1 ml per 10⁷ cells), and incubate at 4°C for 10–20 minutes.  
2.) Wash the cells twice with Staining Buffer.  
3.) Resuspend the cells in Staining Buffer and store at 4°C for up to 72 hours; alternatively, resuspend the cells in 90% FCS/10% DMSO and store at -80°C for long-term preservation.  

## b. Permeabilization of Fixed Cells  
1.) For cryopreserved cells, wash twice to remove DMSO. For cells stored at 4°C, centrifuge to remove the Staining Buffer.  
2.) Resuspend the cells in Wash Buffer and incubate for 15 minutes.  
3.) Centrifuge to remove the supernatant.  
4.) Proceed with intracellular cytokine staining.  

# 6. Intracellular Cytokine Staining  
a. Take an appropriate amount of cytokine fluorescent antibodies or negative controls, dilute them to 50 μl with Wash Buffer. Use this antibody dilution to fully resuspend the fixed and permeabilized cells, and incubate at 4°C in the dark for 30 minutes.  
b. Wash the cells twice with 1× Wash Buffer (250 μl per wash for microplates, 1 ml per wash for test tubes), then resuspend the cells in Staining Buffer and analyze by flow cytometry.