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Intracellular Staining for Flow Cytometry - Protocol & Troubleshooting

Flow cytometry is a technique for the rapid multiparametric analysis of single cells in solution. It has a wide range of applications as a powerful tool in immunology, molecular biology, bacteriology, virology, cancer biology and infectious disease surveillance. It allows the analysis of surface molecules and intracellular antigens at the single cell level. Unlike staining for cell surface markers, staining for intracellular proteins usually requires fixation, permeabilization, and other treatments.

We describe a general staining method for intracellular protein detection using flow cytometry. You can follow the protocol to stain for intracellular antigens while maintaining the morphological characteristics of the cells. In addition to this, Creative Biolabs offers products and services to facilitate the implementation of intracellular staining flow cytometry.

Solutions and Reagents

Stages Solutions and Reagents
Sample Preparation Cell staining buffer, fixation buffer, permeabilization buffer, PBS
Antibody Staining Primary antibody, secondary antibody, antibody dilution buffer, cell staining buffer, PBS

Intracellular Staining for Flow Cytometry Procedure

Intracellular Staining for Flow Cytometry

1. Sample Preparation

Harvest cultured or freshly isolated cells. Prepare single cell suspensions by appropriate treatment of different cell samples and adjust the cell suspension concentration in buffer.

2. Fixation

Fix the cells before performing intracellular staining to ensure the stability of intracellular antigens. The choice of fixative is an important first step, usually using formaldehyde, ethanol, depending on the target antigen and cell type. Add fixative to the cells for 15-20 minutes of incubation. Centrifuge at room temperature and remove the fixation buffer. And wash the fixed cells several times using cell staining buffer.

3. Permeabilization

In order for the antibody to penetrate the cell membrane, the membrane must be permeabilized. Add permeabilization buffer to the fixed cells, mix gently and let stand for 5-10 minutes. Centrifuge at room temperature to remove the supernatant. Then wash the cells several times with the buffer.

4. Antibody Staining

You can choose to do either direct or indirect staining. For direct staining, dilute the primary antibody with buffer and resuspend the cells with the primary antibody solution. Incubate for 15-20 minutes. Then wash with buffer and centrifuge to discard the supernatant.

For indirect staining, after incubation of the primary antibody, centrifuge to remove the supernatant. Dilute the fluorescently labeled secondary antibody with the buffer and resuspend the cell precipitate with the secondary antibody solution. Incubate in the dark for 15-20 minutes. Wash with buffer and discard the supernatant by centrifugation.

Finally, resuspend the cells with cell staining buffer for final flow cytometry analysis.


We provide troubleshooting guides for some common problems in intracellular staining flow cytometry experiments, including possible causes and solutions. The protocols described below are suitable for general applications, and will need to be modified and optimized for any particular sample and specific product. We hope this information will advance your experimental progress.

No or weak signal

  1. Sample causes. First use freshly isolated cells whenever possible, not frozen samples. If using cryopreserved cells, check that the target antigen will not be affected by the freezing/thawing procedure. You will need to adjust the cell population to the recommended density before starting the experiment. Next, check that the target protein is within the cells and present in sufficiently high amounts for detection. For low expression antigens, use the brightest fluorescent dye or two-step staining to increase sensitivity. Finally, remember to perform all protocol steps at 4°C and use cold reagents.
  2. Fixation/permeabilization causes. If no signal is detected, it may be that your target antigen may be inaccessible. Check that the fixation and permeabilization method is correctly used for the target of interest. In addition to carefully selecting the most appropriate fixative and permeabilization reagents, different procedures need to be used depending on the protein being tested. For example, antigens close to the plasma membrane as well as soluble cytoplasmic antigens are suitable for mild cell-penetrating treatments without fixation.
  3. Antibody causes. If your signal is weak, your test antibody may be too dilute. We recommend increasing the amount or concentration of antibody. Or titrate the antibody before use to find the optimal amount for your particular experiment. You can also optimize antibody incubation time and temperature and consider using additional steps to amplify the signal. It is important to use secondary antibodies generated against the species from which the primary antibody was generated.
  4. Fluorescent dye causes. In the selection of fluorescent dyes, it is important to match the target to use. For example, the brightest fluorescent dye is used to detect the lowest density target and the darkest fluorescent dye to detect the highest density target. It is also important to consider the size, conformation, and stability of the fluorescent dye. It is important to control any potential changes to the fluorescence properties during the experiment, such as light exposure, storage time, etc.
  5. Reagent causes. Take care to ensure that all reagents and solutions are of good quality and stored according to the manufacturer's instructions.

High signal

  1. Antibody causes. High antibody concentrations can produce high non-specific binding or very high fluorescence intensity. You can reduce the amount of antibody added to each sample. It is better to titrate the antibody prior to use to find the optimal amount for a particular experiment.
  2. Fluorescent dye causes. Match the appropriate fluorescent dye to the antigen density.
  3. Washing causes. It may occur that unbound antibodies are trapped in the cells. Wash the cells well after each antibody incubation step.

High background

  1. Cell causes. One possibility is due to the presence of dead cells. A reactive dye can be used to exclude the dead cells. The other is due to high autofluorescence. Some cell types may naturally exhibit higher levels of autofluorescence. You can use fluorescent dyes that emit in the red-shift channel or very bright fluorescent dyes to solve the problem.
  2. Antibody causes. Antibody overload causes high background. You can reduce the antibody concentration and use the recommended antibody dilutions to ensure the right dose of antibody. Optimize antibody concentration and incubation time based on cellular protein expression. If possible, avoid using biotinylated antibodies.
  3. Washing causes. Increase the number of washes after staining to ensure that excess antibody is removed.

Unusual scatter profiles

  1. Cell causes. Samples should be fresh and properly prepared. Make sure that the cells in the sample are not lysed and broken. Do not centrifuge cells at high rotor speeds or vortex too vigorously. Perform a sieve on the cells to remove any dead cell debris. Also make sure that the sample is not contaminated with bacteria. Bacteria will automatically fluoresce at low levels. Finally, avoid storing stained cells for long periods of time.

Flow cytometry is one of the most effective methods for analyzing various samples in a short period of time. By intracellular staining it can provide valuable information about the antigen of interest. We are committed to helping our customers achieve better results and hope the above information will serve as a reference guide for your intracellular staining in flow cytometry. If you need further assistance, please contact us.


  1. Adan A, et al. Flow cytometry: basic principles and applications. Critical Reviews in Biotechnology, 2016:163-176.

For research use only. Not intended for any clinical use.

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