Flow Cytometry Protocol for Staining Intracellular Molecules using Detergents 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 and other secreted molecules 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 maintaining the morphological characteristics used to sort the cells. Commonly used detergents include saponin, Triton® X-100, or Tween® 20. 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 is to be performed in the same sample, it is advisable that the surface staining be performed first, since 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)
  • Flow Cytometry Permeabilization Buffer/Wash Buffer I (1X; R&D Systems, Catalog # FC005, or an equivalent solution containing a permeabilization agent such as saponin or Triton X-100)
  • (Optional, see Note in step 3) Flow Cytometry Fixation/Permeabilization Buffer I (1X; Catalog # FC007)
  • 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

Procedure

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

Note: Depending on the specific antibody and cell sample being used, the fixation and permeabilization steps can be performed simultaneously using Flow Cytometry Fixation/Permeabilization Buffer I.

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

Note: Because saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the presence of Permeabilization Buffer I during intracellular staining.

Note: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody should occur now. Add the recommended volume of secondary antibody, 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. Resuspend the cell pellet in 100 – 200 μL of Flow Cytometry Permeabilization/Wash Buffer I.
  4. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 15 minutes at room temperature.
  5. 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.
  6. Wash cells 2 times with Flow Cytometry Permeabilization/Wash Buffer I as in step 3.
  7. Resuspend the cells in 200 – 400 μL PBS (or HBSS) buffer for flow cytometric analysis.

SAMPLE DATA

Figure: Detection of CCL4/MIP 1 beta in Human Blood Monocytes by Flow Cytometry
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Detection of CCL4/MIP 1 beta in Human Blood Monocytes by Flow Cytometry. Human peripheral blood monocytes treated with LPS and Monensin for 5 hours were stained with Mouse Anti-Human CD14 APC conjugated Monoclonal Antibody (Catalog # FAB3832A) and either (A) Mouse Anti-Human CCL4/MIP 1 beta Fluorescein conjugated Monoclonal Antibody (Catalog # IC271F) or (B) Mouse IgG2B Fluorescein Isotype Control (Catalog # IC0041F).To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005).

FACS is a trademark of Becton Dickinson and Company.

Triton is a registered trademark of The Dow Chemical Company.

Tween is a registered trademark of Uniqema Americas LLC.