Name: flow cytometry in cell sorting
Uploaded: 2023-04-30T11:58:15.000Z
Duration: 1 min 23 s
Channel: JoVE

Flow cytometry analyzes a heterogeneous cell population such as white blood cells based on multiple parameters, like size and complexity.
When the suspension is loaded on a flow cytometer, the sheath fluid and vibrating nozzle form single-cell droplets that pass through a laser beam, scattering light in multiple directions.
The forward scatter measuring cell size, and the side scatter indicating cell complexity, are detected through different detector systems.
Plotting forward versus side scatter on a graph helps differentiate cell populations and their proportions in a sample. For instance, the forward and side scatter are higher for granulocytes, which are larger and more granular than lymphocytes.
Fluorescent activated cell sorter or FACS, a specialized flow cytometry, helps analyze and sort cells. It uses fluorescently tagged antibodies specific against certain cellular components -- such as CD4 or CD8 markers on T cells -- added to the sample.
An electrical charging ring adds a charge on single-cell droplets based on the fluorescent tag.
The electromagnets then use this charge to deflect different cell types into separate tubes.

flow cytometry

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
In 1967, Kamentsky and Melamed achieved the diversion of cells away from the flow, allowing cell sorting. But it was not until 1973 that a refined electrostatic sorter, known as a fluorescence assisted cell sorter or FACS, was developed. Typically, the cell suspension passes through a laser beam, and the light scatter, or fluorescence, is detected. The changes in scatter patterns or fluorescence determine how the cells should be sorted.
Applications of Flow Cytometry
Apart from cell counting and sorting, flow cytometry is also used to estimate the DNA and RNA content of cells. When a cell suspension is incubated with fluorochromes, viable cells take up the stains to different degrees based on cell type and growth phase. These differences are detected by the FACS software, which then estimates the relative proportions of the cell types in different growth phases. Since flow cytometry estimates cell parameters such as shape, size, and granularity, it can also be used to differentiate necrotic and apoptotic cells. For example, apoptotic cells are typically more granular and therefore tend to have a higher side scatter of light than the less granular necrotic cells.
Limitations of Flow Cytometry
An obvious limitation of flow cytometry is that it cannot analyze cells that are not suspended in culture, such as those growing on an adherent medium or organized in a tissue. All samples must be dissociated into cell suspensions to generate single-cell droplets; therefore, cell-cell interactions cannot be analyzed. Another constraint stems from the limit on the number of simultaneous detectors. Despite many fluorophores being available, a flow cytometer usually does not have more than 12 detectors to efficiently and consistently detect the different cell subpopulations. While flow cytometry is a powerful technique, it can also generate an overwhelming quantity of data that requires a human expert to analyze and process. And finally, given the high sensitivity of this technique, standard protocols for sample preparation, data recording, and analysis are essentia. However, a lack of standardization of these aspects makes it&#160; difficult to compare results from different studies.

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing ...

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flow cytometry in cell sorting

Flow cytometry in cell sorting tracks cells as they move through a laser beam. The instrument measures light scatter and fluorescence, then uses those signals to separate cells based on their properties. It is also used to count cells and study cell populations in detail.

The method began in 1934 with Andrew Moldavan, a bacteriologist who counted cells in a flowing capillary system. He pushed cells through a capillary tube and viewed them under a microscope. Later, photometry made it possible to measure differently stained cells. In 1965, Louis Kamentsky built the first multiparameter flow cytometer to identify and count cancer cells in cervical tissue specimens.

By 1967, Kamentsky and Melamed had developed a way to divert cells away from the flow, which allowed cell sorting. A refined electrostatic sorter followed in 1973. This device was called a fluorescence assisted cell sorter, or FACS. As cells pass the laser, changes in scatter patterns or fluorescence tell the system how to sort them.

Flow cytometry is not limited to counting and sorting. It can also estimate the DNA and RNA content of cells. When a cell suspension is incubated with fluorochromes, which are fluorescent stains, viable cells take up the stains in different amounts based on cell type and growth phase. FACS software then uses these signals to estimate the relative proportions of cells in different growth stages.

The technique can also help distinguish necrotic and apoptotic cells. Flow cytometry measures cell shape, size, and granularity. Apoptotic cells are typically more granular, so they often show higher side scatter of light than necrotic cells, which are less granular. These differences give researchers another way to compare cell states.

Flow cytometry does have limits. It cannot analyze cells that are not in suspension, such as cells growing on an adherent medium or arranged in a tissue. Samples must first be dissociated into single-cell suspensions, so cell-cell interactions cannot be studied directly. The method is also limited by the number of simultaneous detectors, and most flow cytometers do not have more than 12.

The large amount of data can also be hard to manage. Even with many available fluorophores, a human expert is often needed to analyze and process the results. Because the method is very sensitive, sample preparation, data recording, and analysis must follow standard protocols. When those steps are not standardized, it becomes difficult to compare results across studies.

Flow Cytometry in Cell Sorting

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Flow cytometry in cell sorting tracks cells as they move through a laser beam. The instrument measures light scatter and fluorescence, then uses those signals to separate cells based on their properties. It is also used to count cells and study cell populations in detail. The method began in 1934 with Andrew Moldavan, a bacteriologist who counted cells in a flowing capillary system. He pushed cells through a capillary tube and viewed them under a microscope. Later, photometry made it possible...

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key terms and concepts

Learn how flow cytometry uses laser light, fluorescence, and FACS to count, sort, and analyze cells, plus its key limits in sample handling and data analysis.

Flow Cytometry in Cell Sorting | JoVE K12

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Flow Cytometry in Cell Sorting

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