Flow Cytometry Protocol

Principle

The flow cytometer is an instrument that can detect and sort particles like cells, bacteria, microspheres, and small model organisms. As particles flow individually in front of a light source motivated by a laser transmitter, the signals from those particles can be detected by the detector module and subsequently, transferred to a computer for analysis. Therefore, we can prepare samples that can be measured by flow cytometry by transfection or expression of fluorescent proteins (such as green fluorescent protein), staining with fluorescent conjugated antibodies, or staining with fluorescent dyes (such as propidium iodide). In almost any situation that requires phenotyping a cell population, identifying antigens of interest, or determining the cell cycle status of a cell population, flow cytometry can be a good choice.

Fig. 1 Basic principles of flow cytometry.

Applications of Flow Cytometry

Flow cytometry is mainly applied to basic research in life sciences, especially in microbial analysis, immunology, cell biology (like cell phenotyping), molecular biology (like DNA/RNA analysis, cytokines), and clinical medicine. For example, the application of flow cytometry to measure the number of CD4+ T cells in peripheral blood can be used to monitor the progression of the disease in HIV patients. Flow cytometry can also assist in the diagnosis and classification of various diseases like leukemia.

Fig.2 Partial application of flow cytometry. ¹

Material

Procedure

1. Harvest and wash cells with PBS, and count the total number of cells under a microscope.
2. Resuspend cells in PBS and dilute to 1-5 x 10⁶ cells/mL.
3. Add cell suspension into test tubes, 100μl per tube.
4. Add 0.1-10 μg/mL primary antibody and incubate at 4°C for 60 minutes.
5. Centrifuge at 400g for 5 minutes, wash the cells 3 times and resuspend in ice-cold PBS.
6. Add fluorescent dye-labeled secondary antibody to the cell suspension and incubate at 4°C in the dark for 60 minutes.
7. Centrifuge at 400g for 5 minutes, wash the cells three times and then resuspend in PBS.
8. Analyze cells using flow cytometry.

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Reference

1. Robinson, J. Paul, et al. "Flow Cytometry: The Next Revolution." Cells 12.14 (2023): 1875. Distributed under Open Access license CC BY 4.0, without modification.