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Flow Cytometry Protocol: Intracellular Staining

Intracellular flow cytometry can be used to analyze a variety of intracellular molecules including cytokines, inflammatory mediators, transcription factors, and phosphoproteins. It can provide rich information concerning cellular function and signaling responses. Different from cell surface markers staining, intracellular antigens detection requires cell fixation and permeabilization before staining. Typically, cells are fixed with formaldehyde to preserve the cellular morphology, and then permeabilized with detergent or alcohol. Such fixation/permeabilization treatment allows the antibodies against intracellular antigens across the plasma membrane to stain intracellularly, while maintaining the morphological characteristics of cells.

Notes:

  • For staining of secreted proteins, such as cytokines, a protein transport inhibitor such as Brefeldin A or Monensin, should be added prior to fixation/permeabilization in order to trap the cytokines inside the cells and enable intracellular staining.
  • When surface and intracellular staining is to be performed in the same sample. It is advised that surface staining should be carried out first to avoid any potential effects of the intracellular staining protocol.

Reagents:

  • PBS: Dissolve 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4 and 0.2 g KH2PO4 in 800 mL distilled water. Adjust the pH to 7.4 with HCl and final volume to 1 liter with additional distilled H2O.
  • Cell staining buffer: Add 0.5% BSA and 0.05% Sodium Azide (NaN3) to PBS.
  • Fixation buffer: 1% paraformaldehyde in PBS.
  • Permeabilization buffer: 0.1% saponin in cell staining buffer.

Equipment:

  • Pipettes and pipettors
  • Centrifuge
  • Centrifugal tube

Procedures:

Sample preparation

1. Harvest the tissues and cells, prepare a single-cell suspension and adjust the suspension to a concentration of 1 x 106 cells/mL in cell straining buffer.

Learn more about sample preparation for flow cytometry.

Note: Cell surface staining may be done at this point.

Fixation

2. Add 1 mL fixation solution per 1 x 106 cells and incubate for 15 minutes at room temperature.

3. Centrifuge at 400 x g for 5 minutes at room temperature and remove the fixation buffer.

4. Wash fixed cells with cell staining buffer. Centrifuge at 400 x g for 5 minutes and discard the supernatant.

5. Repeat Step 4 twice.

Permeabilization

6. Resuspend fixed cells in 2 mL permeabilization buffer and centrifuge at 400 x g for 5 minutes at room temperature. Remove the supernatant.

7. Wash the fixed/permeabilized cells with permeabilization buffer. Centrifuge at 400 x g for 5 minutes and discard the supernatant.

8. Repeat Step 7 twice.

Staining

9. Dilute the primary antibody with permeabilization buffer for an optimal working concentration and resuspend the fixed/permeabilized cells with primary antibody solution. Incubate for 15 to 20 minutes at 4°C.

Note: If using primary antibodies directly conjugated with fluorochrome, the incubation should be carried out in the dark, and then skip to Step 14.

10. Centrifuge at 400 x g for 5 minutes at 4°C and remove the supernatant.

11. Wash with permeabilization buffer and centrifuge at 350 x g for 5 minutes. Discard the supernatant.

12. Repeat Step 11 twice.

13. Dilute the fluorescent-conjugated secondary antibody with permeabilization buffer for an optimal working concentration and resuspend the cell pellet with secondary antibody solution. Incubate on ice for 15-20 minutes in the dark.

14. Repeat Step 11 three times

15. Resuspend cells in 200-500 uL cell staining buffer for final flow cytometric analysis.

Browse all the antibodies for flow cytometry.

Learn the principle of flow cytometry.

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