Flow Cytometry Protocol for Staining Intracellular Molecules using Alcohol to Permeabilize the Cell Membrane

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Flow cytometry can be used to analyze various intracellular molecules including phosphorylated signaling proteins and cytokines. Cytokines, small signaling molecules secreted by many cell types, can be detected by flow cytometry in activated cells with the aid of secretion inhibitors, such as monensin or brefeldin A. These compounds prevent the export of newly synthesized proteins by disrupting the ER-Golgi transport machinery. For experimental treatments with stimulation periods of up to 4-6 hours, the secretion inhibitor can be present during the entire incubation period. If the stimulation period is longer than 4-6 hours, the secretion inhibitor should be added for only the last two hours of the incubation.

There are many variables that must be optimized for individual flow cytometry experiments such as antibody incubation time and temperature. Furthermore, to stain intracellular molecules, the cells need to be fixed in suspension and then permeabilized before the detection antibody is added. This fixation/permeabilization treatment allows the antibody to pass through the plasma membrane into the cell interior, while keeping the morphological characteristics used to sort the cells intact. Alcohols, such as methanol or ethanol are commonly used to permeabilize the cell. Cold methanol is typically used as a permeabilization agent when using flow cytometry to detect phosphorylated proteins and transcription factors, because it can increase the reactivity of antibodies to certain nuclear antigens. The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for the staining of intracellular molecules for flow cytometric analysis.

Note: When surface and intracellular staining are to be performed in the same sample, it is advisable that the surface staining be performed first because fixation/permeabilization treatments might decrease the availability of surface antigens. However, take care to avoid using PE or APC conjugates prior to methanol permeabilization. Methanol affects PE and APC, causing a loss of signal.

Please read the protocol in its entirety before starting.

Reagents Required

  • PBS (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4 or Hank’s Balanced Salt Solution (HBSS; 1X)
  • Flow Cytometry Fixation Buffer (R&D Systems, Catalog # FC004, or an equivalent solution containing 1 - 4% paraformaldehyde)
  • -20 °C Methanol
  • Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions)
  • Fluorochrome-conjugated antibodies suitable for use in flow cytometry
  • Isotype Control Antibodies

Materials Required

  • FACS™ Tubes (5 mL round-bottom polystyrene tubes)
  • Pipette Tips and Pipettes
  • Centrifuge
  • Vortex


Note: Staining of surface antigens may be done at this point.

Note: Do not wash excess blocking IgG from this reaction.

Note: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody should occur now. Dilute the secondary antibody in PBS (or HBSS), starting with the concentration suggested in the product datasheet. Incubate for 20-30 minutes in the dark and wash as in step 3.

Note: For a negative control, a separate set of cells should be stained with an isotype control antibody.

  1. Harvest the cells and wash 2 times by adding 2 mL of PBS (or HBSS), centrifuging at 1250-1500 rpm/350-500 x g for 5 minutes, and then decanting buffer from pelleted cells.
  2. Aliquot up to 1 x 106 cells/100 μL into FACS tubes. Add 0.5 mL of cold Flow Cytometry Fixation Buffer and vortex. Incubate at room temperature for 10 minutes. Vortex the cells intermittently in order to maintain a single cell suspension.
  3. Centrifuge cells and decant the Fixation Buffer. Wash the cells 2 times with PBS (or HBSS) as in step 1.
  4. Resuspend the cells in 900 μL of -20 °C methanol. Incubate for 30 minutes at 4 °C.
  5. Centrifuge the cells for 5 minutes at 1250-1500 rpm/350-500 x g. Remove and discard supernatant. Wash 2 times with PBS (or HBSS) as in step 1.
  6. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 15 minutes at room temperature.
  7. Add conjugated antibody (5-10 μL/106 cells or a previously titrated amount) and vortex. Incubate cells for 30 minutes at room temperature in the dark.
  8. Wash cells 2 times with PBS (or HBSS) as in step 1.
  9. Resuspend the cell pellet in 200 - 400 μL of PBS for flow cytometric analysis


Figure: Detection of STAT6 in Human peripheral blood mononuclear cells (PBMCs) by Flow Cytometry
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Detection of STAT6 in Human peripheral blood mononuclear cells (PBMCs) by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) either (A) Th2-stimulated or (B) unstimulated were stained with Rabbit Anti-Human Phospho-STAT6 (Y641) Monoclonal Antibody (Catalog # MAB3717) followed by anti-Rabbit IgG PE-conjugated secondary antibody (Catalog # F0110) and Mouse Anti-Human CD4 APC-conjugated Monoclonal Antibody(Catalog # FAB3791A). Quadrant markers were set based on control antibody staining(Catalog # MAB1050). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with methanol.

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