Intracellular Flow Cytometry Protocol Using Alcohol (Methanol)

Contents 

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

Materials

• 1 X PBS (0.137 M NaCl, 0.05 M NaH₂PO₄, pH 7.4)
• Bovine Serum Albumin to make 1% BSA in PBS
• -20 °C Methanol (keep on ice during procedure)
• Intracellular Staining Kit (Catalog # NBP2-29450) which includes 10X Permeabilization Buffer, 1000X Brefeldin A (inhibitor), 1X Fixation Buffer, and 1X Staining Buffer or equivalent products
• Deionized water to dilute stock Permeabilization Buffer to 1X
• Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions)
• Primary Antibodies
• Isotype Control 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

After removing media from adherent cells, add cold PBS to remove residual growth factors from cell culture media.

• Harvest cells with a 1% BSA solution in PBS and then proceed with washing in Step 2.
• Adherent cell lines may require 0.5 mM EDTA to facilitate removal and then washed according to Step 2. Exposure time with EDTA should be minimal.

To prepare tissues for flow cytometry, mechanical and/or enzymatic disaggregation is required.

• First, mince the tissue into small sections that expose the cells and suspend in PBS. Enzymatic digestion may be required after mincing the tissue, but digestion buffer will be tissue type dependent.
• Next, pass the minced tissue suspension through a fine gauge needle several times until all cells are fully in suspension. If you experience resistance, exchange needle with a larger gauge to dissociate cells first.

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, create a 1:1 Trypan Blue exclusion stain to count cells using a hemocytometer.
   • Tip: Staining of surface antigens may be done at this point. Cell viability staining should be performed prior to cell fixation.


4. Add 500 μL of cold 1X Fixation Buffer into FACS tubes required for your experiment. Aliquot up to 1 x 10⁶ cells/100 μL (i.e. 5 x 10⁶ cells) and vortex to mix. A separate set of cells should be stained with an isotype control antibody as a negative control.
5. Incubate the cells at room temperature for 10 minutes. Gently vortex intermittently to maintain a single cell suspension.
6. Add 2 mL of PBS. Centrifuge cells at 1,300 RPM and 4°C for 5 minutes. Decant the Fixation Buffer.
7. Resuspend cell pellet in 900 μL of cold methanol and incubate for 30 minutes at 4°C. Gently vortex intermittently to maintain a single cell suspension.
8. Centrifuge cells at 1,300 RPM and 4°C. Decant the methanol.
9. Remove methanol remnants with 2 mL of PBS. Centrifuge at 1,300 RPM (500 x g maximum) and 4°C for 5 minutes. Discard supernatant.
10. Resuspend each cell pellet in 150 μL of 1X Permeabilization Buffer. Add 1 μg blocking IgG per 1 x10⁶ cells and let stand for 15 minutes at room temperature. Do NOT wash excess blocking IgG from this reaction and keep cells in the presence of Permeabilization Buffer during intracellular staining.
11. Add 5-10 μL of conjugated antibody (or a previously titrated amount) per 1 x 10⁶ cells and vortex. Incubate cells for 30 minutes at room temperature protected from light. Gently vortex intermittently to maintain a single cell suspension.
12. Wash cells using 2 mL of Permeabilization Buffer. Centrifuge at 1,300 RPM (500 x g maximum) and 4°C for 5 minutes. Decant the supernatant.
13. Resuspend cells in 200 – 400 μL of 1X Staining Buffer for final flow cytometric analysis.

Flow Cytometry Basics

Why Use Flow Cytometry?

In addition to phenotyping cells according to cellular morphology through forward and side scatter, antigen expression can be examined in various cell subtypes using flow cytometry. To assess intracellular antigen expression, cells must be fixed in suspension and then permeabilized before adding detection antibodies. It is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. Using antibodies for various intracellular molecules including phosphorylated signaling proteins and cytokines, flow cytometry is a powerful high-throughput tool. Several parameters can be measured simultaneously on millions of individual cells through the combination of different fluorescent probes in a multicolor panel. Permeabilization allows an antibody to pass freely across the membrane while maintaining the morphological characteristics required for phenotypic analysis and sorting. Due to their large size, tandem dyes are not recommended for intracellular staining as they have difficulty crossing the cell membrane to reach intracellular target proteins.

When to Use Alcohols to Permeabilize

Organic solvents (i.e. methanol/acetone) are more vigorous surfactants that can be used to permeabilize membranes by dissolving membrane lipids. Detergents on the other hand, create a porous membrane through mild permeation instead of dissolution of the membrane. Cold methanol is typically used to detect phosphorylated proteins and transcription factors. This flow cytometry protocol using alcohol as a permeabilization reagent is often required when detergents produce high background staining. It is important to note that methanol may decrease PE and APC conjugate signals. Therefore, cells should be permeabilized and washed prior to antibody addition to minimize methanol-fluorochromes interaction.

Other Considerations

• When both intracellular and extracellular staining are required, it is advised to perform the extracellular surface staining first. Reagents used for fixation and permeabilization may decrease surface antigen availability.
• Cell viability staining is highly recommended if using tissue. Both mechanical and enzymatic digestion will cause some cell damage and can result in non-specific binding.
• Cytokines and other secreted molecules can be detected by flow cytometry in activated cells with the aid of secretion inhibitors, such as Brefeldin A. These inhibitors prevent intracellular protein transport and are required for staining secreted proteins. These compounds prevent the export of newly synthesized proteins by disrupting the ER-Golgi transport machinery, ultimately trapping the proteins in their respective cellular compartments. For experimental treatments with stimulation periods of up to 4 hours, the secretion inhibitor can be present during the entire incubation period. If the stimulation period is longer than 6 hours, the secretion inhibitor should be added for only the last 2 hours of the incubation.