Intracellular Nuclear Target Flow Cytometry Protocol Using Detergents

The following flow cytometry protocol for staining intracellular nuclear targets using detergents to permeabilize cell membranes has been developed and optimized by Bio-Techne. Individual experimental designs for flow cytometry must be optimized, including antibody dilution and incubation time but it is recommended to use 1 x 10⁶ cells per 100 μL of sample. For low cell density or poorly expressed intracellular targets, techniques like Single-Cell Westerns may be advantageous. Learn more about the benefits of Milo™ , our Single-Cell Western platform. 

Please read the protocol in its entirety before starting.

Contents

• Materials
• Sample Preparation
• Methods
• Why Use Flow Cytometry?
• Permeabilizing the Nuclear Membrane
• Other Considerations

Materials

• 1 X PBS (0.137 M NaCl, 0.05 M NaH₂PO₄, pH 7.4)
• Flow Cytometry Fixation Buffer (Catalog # FC-004) or an equivalent solution containing 1 - 4% paraformaldehyde
• Permeabilization Buffer: 0.1–1% Triton or NP-40 in PBS
• Flow Cytometry Staining Buffer (Catalog# 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 (Catalog # NBP2-31156); Propidium Iodide (Catalog # NBP2-31155); or 7-AAD (Catalog # 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 to wash the cells. Centrifuge at 1,300 RPM (500 x g maximum) and 4 °C for 5 minutes, decanting the supernatant. Wash cells three times.
3. Using a small aliquot, count the cells. Using a hemocytometer and a 1:1 Trypan Blue exclusion stain, count cells to determine cell viability before starting.
   • Tip: Surface and cell viability staining should be performed now. 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. A separate set of cells should be stained with an isotype control antibody as a negative control.
5. After adding the cells to 500 μL of cold Fixation Buffer per sample tube, vortex to mix. Incubate the cells at room temperature (RT) for 10 minutes.
6. Centrifuge cells at 1,300 RPM and 4°C. Decant the Fixation Buffer.
7. Resuspend each cell pellet in 150 μL Permeabilization buffer (0.1–1% Triton or NP-40 in PBS). Add 1 μg blocking IgG per 1 x 10⁶ cells and let stand for 15 minutes at RT. Do NOT wash excess blocking IgG from this reaction. It is important to keep cells in Permeabilization Buffer during intracellular staining.
8. Add 5-10 µL of conjugated antibody (or a previously determined 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 4 °C protected from light.
9. Add 2 mL of Permeabilization Buffer to wash cells ONCE. Centrifuge at 1,300 RPM (500 x g maximum) and 4 °C for 5 minutes, decanting the supernatant.
   • 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 the cell pellet in 150 μL of Permeabilization Buffer. Add the recommended volume of secondary antibody and incubate for 30 minutes at 4°C protected from light. Gently vortex intermittently to maintain a single cell suspension. Wash cells ONCE using Permeabilization Buffer 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 can be used to characterize single-cell suspensions. Cells can be identified according to morphological phenotypes through forward and side scatter, while antigen expression can be examined in various cell subtypes through use of antibodies in applications like flow cytometry. To assess intracellular antigen expression, cells must be fixed in suspension and then permeabilized prior to adding detection antibodies. Through permeabilization, antibodies can freely pass across the cell membrane.

Permeabilizing the Nuclear Membrane

Triton or NP-40 at a concentration of 0.1–1% in wash buffer is a suitable Permeabilization Buffer for nuclear antigen staining. These detergents dissolve the nuclear membrane completely, allowing antibodies to interact with nuclear envelope and nuclear matrix targets. Due to a cell’s ability to regenerate its cell membrane, it is important to keep cells in Permeabilization Buffer during intracellular staining to prevent the reversal of permeabilization.

Other Considerations

• When both intracellular and extracellular staining are to be performed, it is advised to perform the extracellular surface staining first, as the reagents used for fixation and permeabilization tend to decrease surface antigen availability. Tandem dyes are not recommended for intracellular staining protocols due to their inability to cross the cell membrane.
• If you experience a lot of non-specific binding background signal when using detergents, consider using alcohol permeabilization that may allow for better nuclear antigen interaction.
• After cell fixation, centrifugation speeds can be slightly increased for better yields. In general, choose a centrifugation speed at which cells can be sufficiently washed, allowing for removal of the supernatant with minimal cell loss but still allows for cell resuspension.