Intracellular Flow Cytometry Protocol Using Detergents

Flow Cytometry Protocol for Intracellular Targets Using Detergents

The following flow cytometry staining protocol for intracellular molecules using detergents to permeabilize cell membranes has been developed and optimized by Bio-Techne. For best results, use 1 x 10⁶ cells per 100 μL of sample. Individual experimental designs for flow cytometry must be optimized, including antibody dilution and incubation time. For low cell density or poorly expressed intracellular targets, techniques like Single-Cell Westerns may be advantageous. Learn more about the benefits of Milo^™, a Single-Cell Western platform. Please read the staining protocol in its entirety before starting.

  Download our Flow Cytometry Protocol

CONTENTS

• Materials
• Sample Preparation
• Methods
• Why Use Flow Cytometry
• When to Use Detergents
• Other Considerations

Materials

• 1 X PBS (0.137 M NaCl, 0.05 M NaH₂PO₄, pH 7.4)
• Flow Cytometry Fixation Buffer (R&D Systems, Cat No. FC-004) or an equivalent solution containing 1 - 4% paraformaldehyde
• Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems, Cat No. FC-005) or an equivalent solution containing a permeabilization agent such as Saponin, Triton^® X-100 or Tween^® 20 (0.1-0.5% in your wash buffer).
• Flow Cytometry Staining Buffer (R&D Systems, Cat No. FC-001) or an equivalent solution containing BSA and sodium azide
• Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions)
• Primary Antibodies
• Isotype Control Antibodies
• Optional: Secondary Antibodies
• Recommended viability dye: DAPI, Novus Biologicals, Cat No. NBP2-31156; Propidium Iodide, Novus Biologicals, Cat No. NBP2-31155; or 7-AAD, Novus Biologicals, Cat No. NBP2-29446
• Trypan Blue
• FACS Tubes (5 mL round-bottom polystyrene tubes)
• Pipettes with appropriate tips
• Centrifuge
• Vortex

Sample Preparation

Methods

1. Harvest your cells (see Sample Preparation for guidance).
2. Add 2 mL of PBS with a pipette. Centrifuge at 1,300 RPM (500 x g maximum) and 4°C for 5 minutes, decanting the supernatant. Wash 3 times.
3. Using a small aliquot, count cells in a 1:1 Trypan Blue exclusion stain using a hemocytometer before starting.


Tip: Aliquot up to 1 x 10⁶ cells per 100 μL in Flow Cytometry Staining Buffer if performing cell surface staining. Cell viability dyes must be introduced before fixation and should be done at this point. If only nuclear staining is performed, proceed with cell fixation.


4. Add 500 μL of cold Fixation Buffer into FACS tubes required for your experiment. Aliquot up to 1 x 10⁶ cells/100 μL and vortex to mix. Incubate the cells at room temperature for 10 minutes. Gently vortex intermittently to maintain a single cell suspension. A separate set of cells should be stained as a negative control.
5. Centrifuge cells at 1,300 RPM and 4°C. Decant the Fixation Buffer.
6. Wash cells ONCE using 2 mL of PBS. Centrifuge at 1,300 RPM (500 x g maximum) and 4°C for 5 minutes. Decant the supernatant.
7. Resuspend cell pellets in 150 μL of Flow Cytometry Permeabilization/Wash Buffer I. Add 1 μg blocking IgG per 1 x 10⁶ cells and let stand for 15 minutes at room temperature. Do NOT wash excess blocking IgG from this reaction. It is important to keep the cells in the presence of Permeabilization/Wash Buffer I during intracellular staining.
8. Add 5-10 µL of conjugated antibody (or a previously titrated amount) per 1 x 10⁶ cells and vortex. For unconjugated antibodies, be sure to check the data sheet for any appropriate concentrations validated for use in flow cytometry. 1 µL of primary antibody per 1 x 10⁶ cells is a good starting point. Incubate cells for 30 minutes at room temperature in the dark.
9. Wash cells ONCE with 2 mL of Flow Cytometry Permeabilization/Wash Buffer I. Centrifuge suspension at 1,300 RPM (500 x g maximum) and 4°C for 5 minutes.


Tip: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody is required. After washing cells to remove the primary antibody, resuspend cell pellets in 150 μL of Flow Cytometry Permeabilization/Wash Buffer I. Add the recommended volume of secondary antibody and incubate for 30 minutes protected from light. Gently vortex intermittently to maintain a single cell suspension. Wash cells ONCE using Flow Cytometry Permeabilization/Wash Buffer I instead of PBS.


10. Resuspend the cell pellet in 200 – 400 μL of Flow Cytometry Staining Buffer for flow cytometry analysis.

Flow Cytometry Basics

Why Use Flow Cytometry

Flow cytometry is a powerful, high-throughput technology that is used to characterize single-cell suspensions. In addition to phenotyping cells in suspension according to cellular morphology through forward and side scatter, antigen expression can be examined in various cell subtypes using flow cytometry. By combining different fluorescent probes in a multi-color panel, several parameters can be measured simultaneously on millions of individual cells. Flow cytometry can be used to analyze various intracellular molecules including phosphorylated signaling proteins and cytokines. To assess intracellular antigen expression, cells must be fixed in suspension and then permeabilized before adding antibodies. This permeabilization step allows the antibody to pass freely through the plasma membrane while maintaining the morphological characteristics required for phenotypic analysis and sorting. It is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. The large size of tandem dyes impede membrane transport thus are not recommended for intracellular staining.

When to Use Detergents to Permeabilize

Commonly used detergents include Saponin, Triton^® X-100 or Tween^® 20 at a concentration of 0.1-0.5% in PBS. These mild detergents are suitable for cytoplasmic antigens and enable antibodies to permeate the plasma membrane without dissolving the membrane completely. Due to a cell’s ability to regenerate its membrane, cells must be kept in Permeabilization Buffer I to prevent the loss of membrane permeabilization while staining for intracellular antigens. More vigorous detergents such as 0.1–1% Triton or NP-40 in PBS are better for nuclear antigen staining due to their ability to dissolve nuclear membranes.

Other Considerations

• When both intracellular and extracellular staining are to be performed, it is advised to perform extracellular surface staining first as the fixation and permeabilization process decreases surface antigen availability.  Depending on the cell type, the cell fixation and permeabilization step can be performed simultaneously using Flow Cytometry Fixation/Permeabilization Buffer I (R&D Systems, Cat No. FC007). 
• When assessing antigens close to the plasma membrane or soluble cytoplasmic antigens, mild cell permeabilization without fixation may be required.
• Using detergents can result in high background staining.  If needed, alcohol permeabilization is a good alternative approach for intracellular targets.
• Heparin can stimulate white blood cells to release cytokines, so if measuring intracellular cytokines consider using EDTA coated collection tubes when using whole blood cell sample types.

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Intracellular staining for IFN γ using a saponin-based permeabilization buffer. Human PBMCs were stimulated with a restimulation cocktail (Tocris Bioscience Catalog # 5476/1ML) for three hours per the manufacturer’s recommendation. Cells were then harvested and stained with a fixable viability dye prior to cell surface staining with CD3 AF700 (R&D Systems Catalog # FAB100N), CD45RA AF488 (R&D Systems Catalog # FAB1430G) and Ms IgG2a AF647 isotype control antibody (R&D Systems Catalog # IC003R) (a) or isotype matched CD69 AF647 antibody (R&D Systems Catalog # IC0041R) (b). Cells were then washed, fixed with 1% paraformaldehyde and permeabilized prior to staining with a Ms IgG2b PE isotype control antibody (a) or isotype matched IFNγ PE antibody (b).