2.1 Intracellular Factor Staining

12 Feb 2026 by ma kun

Intracellular Factor Staining Protocol

Classification of Target Antigens

Three antigen categories for flow cytometric detection:

  • Surface antigens: CD3, CD4, CD8, etc.
  • Cytoplasmic intracellular antigens: IL-4, IFN-γ, IL-17, Granzyme B, Perforin, etc.
  • Nuclear intracellular antigens: Transcription factors including Foxp3, RORγ, Oct3/4


Table 1. Recommended protein transport inhibitors for human and mouse assays
Species Target Cytokines & Chemokines Inhibitor Selection
Human IL-1α, IL-6, IL-8, TNF-α Monensin
Human IFN-γ, IL-2, IL-10, IL-12, MCP-1, MCP-3, MIG, MIP-1α, RANTES Monensin or Brefeldin A
Mouse IL-6, IL-12, TNF-α Brefeldin A
Mouse GM-CSF, IL-3, IL-4, IL-5, IL-10 Monensin
Mouse IFN-γ, IL-2 Monensin or Brefeldin A

1. In Vitro Cytokine Stimulation

Multiple protocols induce cellular cytokine secretion. Polyclonal activators are widely used for robust cytokine production, including: PMA plus calcium ionophore/ionomycin, phytohemagglutinin (PHA), staphylococcal enterotoxin B (SEB), and anti-TCR/CD3 monoclonal antibodies (with or without co-stimulatory anti-CD28).




Figure 1. Inhibitory Mechanism of Activated T Cells

 

Notes

  • PMA alone downregulates surface CD4 on mouse T cells.
  • Combined PMA + calcium ionophore causes stronger CD4 loss, plus reduced CD8 expression on mouse thymocytes and peripheral T cells from both humans and mice.

2. Sample Preparation

1) Cell Preparation

Treat ex vivo tissue or cultured cells with protein transport inhibitors to retain intracellular cytokines. Isolate cells, resuspend in staining buffer, count, and aliquot into tubes or microplates for immunofluorescent staining. Protect all samples from light during incubation and storage.

2) Fc Receptor Blocking

Fc block reagents minimize non-specific antibody binding.

  1. For mouse samples: Use purified 2.4G2 anti-FcγII/III antibody to block Fc-mediated background staining.
  2. Resuspend 1×106 cells in 100 μL staining buffer, add 1 μg FcBlock, incubate 15 min at 4°C.
  3. Wash cells, then add pre-diluted fluorophore-conjugated antibodies targeting surface antigens.

3. Cell Surface Staining

  1. Resuspend 1×106 cells in 50 μL staining buffer; add optimized fluorophore-conjugated mAbs (e.g., CD3, CD4, CD14, CD19). Incubate 30 min at 4°C.
  2. Wash twice with staining buffer: 250 μL per microplate well, 1 mL per tube. Centrifuge at 250×g after each wash.
  3. For human cells: Pre-incubate with human serum or excess homologous irrelevant Ig, plus isotype control antibodies to block Fc receptors.

4. Cell Fixation & Permeabilization

  1. Thoroughly resuspend cells, add fixation/permeabilization buffer (100 μL per well, 250 μL per tube). Incubate 20 min at 4°C.
  2. Wash twice with 1× wash buffer (250 μL per well, 1 mL per tube).

Notes

  • Vortex thoroughly before adding fixation/permeabilization buffer to prevent cell clumping.

5. Intracellular Cytokine Staining

  1. Dilute cytokine fluorophore antibodies and corresponding negative isotype controls to 50 μL total volume with wash buffer. Resuspend fixed/permeabilized cells fully in antibody mix, incubate 30 min at 4°C in the dark.
  2. Wash twice with 1× wash buffer (250 μL per well, 1 mL per tube). Resuspend final cell pellets in staining buffer for flow cytometric acquisition.
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