Flow Cytometry Using Methanol Permeabilization

Licia Miller   Product Manager

Flow cytometry is a powerful technique that is often used to analyze surface and internal antigens of cells. When detecting intracellular antigens, cells usually need to be fixed and permeabilized. Methanol permeabilization is a commonly used treatment method that uses ice-cold methanol solution to effectively fix cells and disrupt cell membranes, allowing antibodies to enter the cell and bind to target antigens. This method is suitable for a variety of cell types, especially experiments that require the detection of antigens inside organelles such as the nucleus and mitochondria.

The methanol permeabilization method has several significant advantages. For example, methanol can quickly fix cells and destroy the cell membrane and nuclear membrane, making it easier for antibodies to enter the cell. Secondly, the cells treated with methanol can be stored at -20°C for a long time, which is convenient for subsequent experimental arrangements. In addition, the methanol permeabilization method is suitable for the detection of a variety of intracellular antigens, especially for some modified antigens such as phosphorylated proteins that are difficult to detect by other methods. However, it should be noted that methanol may have an effect on certain antigenic epitopes, so small-scale experimental verification is required before use.

Through the methanol permeabilization method, researchers can efficiently perform flow cytometric analysis of intracellular antigens while maintaining the integrity of cell structure, providing strong support for cell biology research.

Experimental Steps

1. Collect and Fix Cell Samples

1.1 Cultivate cells to an appropriate growth stage and collect the cells. Adherent cells must first be digested with trypsin to prepare a single-cell suspension.

1.2 Centrifuge to allow single cells to settle at the bottom of the tube and discard the supernatant.

Note: Centrifugation conditions need to be determined according to the cell type and reagent volume. Generally, 150-300 Centrifuge at 4°C for 1-5 min.

1.3 Add an appropriate amount of 4% formaldehyde solution to resuspend the cells. Generally, add 100 μl for every 106 cells. Pipette thoroughly to mix well to fix the cells and leave at room temperature for 15 minutes.

1.4 Centrifuge and discard the supernatant.

1.5 Resuspend cells with 1 × PBS to remove excess formaldehyde.

1.6 Centrifuge and discard the supernatant, which can be used for subsequent membrane permeabilization operations.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. Such antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and should not be added prior to permeabilization. If you are unsure, perform a small-scale experiment.

2. Cell Permeabilization

2.1 Add 90% ice-cold methanol (100% methanol can be prepared using 1 × PBS was used to dilute the fixed cells.

2.2 Incubate on ice for at least 10 minutes.

2.3 Centrifuge and discard the supernatant.

2.4 Use sufficient amount of 1 × PBS to wash cells and centrifuge to remove methanol. Repeat the washing process as necessary.

3. Immunostaining

3.1 Prepare primary antibody dilution according to the recommended antibody dilution.

3.2 Resuspend cells with 100 µl primary antibody dilution and incubate at room temperature for 1 hour.

3.3 Wash the cells with 1 × PBS, then centrifuge, discard the supernatant, and repeat 1 to 2 times.

3.4 If using directly conjugated antibodies, resuspend the cells in 200-500 µl 1 × PBS for analysis on a flow cytometer.

If using an unconjugated primary antibody, prepare the fluorescently conjugated secondary antibody dilution in advance.

3.5 Resuspend the cells with 100 µl of secondary antibody dilution and incubate at room temperature for half an hour.

3.6 Wash the cells with 1 × PBS, then centrifuge，discard the supernatant, and repeat 1 to 2 times.

3.7 Resuspend the cells in 200-500 µl 1 × PBS for analysis on a flow cytometer.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/