Flow Cytometry Protocol

Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for flow cytometry assays.

Flow cytometry is the detection of proteins through the use of fluorescently labeled antibodies. It can detect multiple proteins at the same time and analyze individual cells. At the same time, flow cytometry detection is characterized by high speed, high precision, and good accuracy. These features make flow cytometry-based detection of proteins a common method for detecting proteins. For the detection of proteins by flow cytometry, Creative Biolabs has summarized the perfect flow cytometry protocol to help your research.

Preparation

1. Any centrifuge-compatible container
2. Suspension/washing buffer (PBS, 5-10% fetal cell serum (FCS))
3. Optional - red blood cell lysis buffer
4. Cell suspension
5. Viability dye
6. Fixative (e.g., 1-4 % paraformaldehyde, 90% methanol, or acetone)
7. Permeabilization solution (for example, Triton X-100, NP-40, or Saponin)
8. The choice of materials will depend on the type of cells analyzed and, if applicable, the secondary antibody used.
9. FcR blocking buffer
10. Conjugated primary antibody
11. Primary antibody
12. Conjugated secondary antibody

Procedure

Live/dead staining with vitalizing dyes

Since dead cells tend to produce non-specific binding antibodies, we must exclude these cells from the analysis. Reactive dyes allow us to differentiate between live and dead cells and exclude dead cells from the data collection and analysis process.

1. Staining of cells with a viability dye (generally, incubate cells with dye in the dark at 4°C).
2. Cells are washed twice with washing buffer. Centrifuge the cells at 200 g at 4°C for 5 min to resuspend the cells.
3. Blocking is performed when detecting extracellular targets, and immobilization and permeabilization are performed when detecting intracellular targets.

Fixation and permeabilization (only for intracellular staining)

When staining intracellular targets, we must perform additional fixation and permeabilization steps. Fixation is required to maintain the structure of the intracellular protein. Permeabilization disrupts the cell membrane, allowing the antibody to enter the cell and stain the intracellular target.

Tips:

Antigens close to the plasma membrane and soluble cytoplasmic antigens require slight cell permeabilization without fixation.

Cytoskeletal antigens, viral antigens, and some enzyme antigens usually work best when fixed using high concentrations of acetone, alcohol, or formaldehyde.

Antigens in cytoplasmic organelles and granules require different fixation and permeabilization methods, depending on the antigen.

Epitopes need to remain accessible.

1. Immobilization of cells in the fixative of your choice: Cells are spun until precipitated (200 g, 5 min, 4°C), the supernatant is discarded, and the precipitate is resuspended in fixative. Incubate cells with fixative.

Fixative	Procedure
1-4% paraformaldehyde (PFA)	15-20 min on ice
90% methanol	10 min at -20°C
100% acetone	10-15 min on ice

2. Cells are washed twice with suspension buffer: Cells are spun until precipitated (200 g, 5 min, 4°C), the supernatant is discarded, and the pellet is resuspended in wash buffer.
3. Permeabilize the cells by incubating them with a suitable detergent: Cells are spun until precipitated (200 g, 5 min, 4°C), the supernatant is discarded, and the precipitate is resuspended in detergent solution. Incubate the cells in detergent for 10–15 minutes at room temperature (This step is not necessary if acetone is used as a fixative, as acetone also permeabilizes the cells).
4. Cells are washed twice with suspension buffer: Cells are spun until precipitated (200 g, 5 min, 4°C), the supernatant is discarded, and the precipitate is resuspended in wash buffer.
5. Proceed to blocking.

Blocking

Blocking proteins and Fc domains is essential to prevent the non-specific binding of antibodies to cells.

1. Fc receptors are blocked with a blocking buffer: Incubate the cells for 30-60 min at 4°C in the dark with one of the following buffers.
2. Cells are washed twice with wash buffer: Spin the cells downward (200 g, 5 min, 4°C), remove the supernatant, and resuspend the precipitate after each wash.
3. Proceed to antibody incubation.

Antibody incubation

Cells are stained by conjugated antibodies with fluorophores for indirect or direct detection in flow cytometry.

Direct antibody labeling

1. Dilute the conjugated primary antibody in suspension buffer.
2. Incubate cells in pre-diluted primary antibody: Centrifuge cells to pellet (200 g, 5 min, 4°C), discard supernatant and resuspend cells in primary antibody solution. In addition, incubate at 4°C for 20-30 min in the dark. Fixed cells can be incubated at room temperature or 4°C.
3. Wash the cells twice with suspension buffer: Spin the cells down (200 g, 5 min, 4°C), remove the supernatant, and then resuspend the pellet after each wash.
4. As soon as possible detect the sample in a flow cytometer. (If the cells cannot be analyzed immediately after staining and are not fixed earlier, the stained cells can be fixed at this step, i.e., 1-4% PFA, 20 min at 4°C.)

Indirect antibody labeling

1. Dilute primary and secondary antibodies in suspension buffer.
2. Incubate cells in pre-diluted primary antibody: Cells are spun into a pellet (200 g, 5 min, 4°C), the supernatant is discarded, and the pellet is resuspended in primary antibody solution. In addition, incubate at 4°C for 20-30 min in the dark. Fixed cells can be incubated at room temperature or 4°C.
3. Wash the cells twice with suspension buffer. Centrifugal cell with 200 g, 5 min, 4°C to harvest cell pellet.
4. Incubate cells in pre-diluted secondary antibody: Centrifugal cell with 200 g for 5 min, and the pellet is resuspended in the secondary antibody solution. Incubate the cells in the dark for 20-30 min.
5. Wash the cells twice with wash buffer. Centrifugal cell with 200 g for 5 min, and the pellet is resuspended in the wash buffer.

As soon as possible detect the sample in a flow cytometer. (If the cells cannot be analyzed immediately after staining and are not fixed earlier, the stained cells can be fixed at this step, i.e., 1-4% PFA, 20 min at 4°C.)

Detection and data analysis

After antibody incubation, we can perform the experiment on a flow cytometer. The procedure is highly dependent on the equipment used, so always consult the manufacturer first.

When surface-stained cells are live and not fixed or permeabilized, they can be separated using fluorescence-activated cell sorting (FACS). Using FACS, live cells can be divided into different populations based on their characteristics. We can then perform downstream analysis on the isolated cells.

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