Flow Cytometry Definition

While laboratories all around the globe are working to improve flow cytometry protocols, they usually consist of the following steps:

1. Formaldehyde is used to immobilise the proteins of interest and their transitory signalling events in cells.

2. Methanol or detergent are added to the test tube to render the cells permeable to antibodies, allowing antibodies to access their intracellular regions.

3. Fluorescently tagged antibodies are added, and they must be carefully chosen to provide optimum epitope targeting and antigen detection while staining numerous surface and intracellular proteins at the same time.

4. The test tube is put in the flow cytometer, and one cell at a time, the fluid is permitted to enter – and then depart – the flow chamber.

5. The light that bounces off each cell as it traverses the laser beam is sent to light/color detectors.

6. After the data from this experiment has been analysed, the unique characteristics of the cells and their cell signalling patterns may be discovered.

Antibody staining can also be different:

1. Direct staining involves incubating cells with an antibody that has been directly coupled to a fluorochrome (e.g., FITC). This is a one-step incubation technique that works well for intracellular staining.

2. In indirect staining, the primary antibody is not labelled, but a fluorochrome-labeled secondary antibody is used to detect it. This technique allows researchers to produce unconjugated primary antibodies against a wide range of targets, expanding their target protein options.

3. A stain of intracellular antigens is referred to as intracellular staining.

4. Finally, a Golgi block can be used to tag and track proteins produced by a cell, followed by intracellular labelling.