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

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.

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
  • Detection Antibodies
  • 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: 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 300 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 at 300 x g for 5 minutes and decant Fixation Buffer. Wash cells 2 times with PBS (or HBSS) as in step 1. Resuspend the cells in 900 μL of -20 °C methanol. Incubate for 30 minutes at 4 °C.
  4. Centrifuge the cells for 5 minutes at 300 x g. Remove and discard supernatant. Wash 2 times with PBS (or HBSS) as in step 1.
  5. Add 10 μL of conjugated antibody (or a previously titrated amount) and vortex. Incubate cells for 30 minutes at room temperature in the dark.
  6. Wash cells 2 times with PBS (or HBSS) as in step 1.
  7. Resuspend the cell pellet in 200 - 400 μL of PBS for flow cytometric analysis.


Figure: Detection of phospho-ERK1/ERK2 by Flow Cytometry
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Detection of Phospho-ERK1/ERK2 by Flow Cytometry. The Jurkat T cell line was unstimulated (light orange; open histogram) or treated with 50 μg/mL PMA for 10 minutes (dark orange; filled histogram). Cells were incubated with the Anti-Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) Monoclonal Antibody (Catalog # MAB1018) or an isotype control antibody (Catalog # AB105C; blue; open histogram), followed by staining with a Fluorescein-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0112). The cells were fixed in paraformaldehyde and permeabilized with ice cold methanol.

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