The Science behind the detection and measurement of tiny and live cells!
The method of flow cytometry has been a boon to researchers worldwide. It is popular among the researchers as it utilizes light to count the cells in a heterogeneous mixture and does their profiling. Flow cytometry is a rapid and accurate technique that is powerful enough to record the data from the heterogeneous mixture containing the live cells.
It’s been a while I have to sharpen my knowledge of Flow Cytometry. So, I took a day to read the basics and many journals and I wondered what if I can simplify the technique of Flow Cytometry for you all and make it easy for understanding.
What do we understand by Flow Cytometry?
The fields of life sciences and biomedical sciences use this cell biology technique widely. Moreover, flow cytometry can be used in every framework where the researcher requires frequent analysis of loose cells of a large population in liquid media. Furthermore, flow cytometry is a useful technique in immunology to identify, segregate, and characterize various immune cell types by the purity of the morphology and size.
When any other detail is required antibodies are tagged with fluorescent dyes, and lifted against the specific cell surface antigen and can be used to better recognize and isolate specific sub-populations within a larger group in flow cytometry. Using a flow cytometer machine, cells or other particles present in a liquid stream are passed through a laser light beam in single file fashion, and interaction with the light is measured by an electronic detection apparatus as light scatter and fluorescence intensity.
Basic Principle of Flow Cytometry
- The sample to experiment is passed through a small channel one at a time.
- Light brightens the cells in the channel.
- It is fitted with a series of sensors that diagnose the types of light that are refracted or emitted from the cells.
- The data hence acquired by the sensors are assembled and homogenized to build an inclusive picture of the sample.
General Experiment Method of Flow Cytometry
- The first and basic step for Flow cytometry is Sample preparation.
- While we are preparing the sample it is very important to keep in mind that the sample must be in single cell suspension in order to avoid clogging up and then we can proceed with the analysis.
- Addition of fluorophore antibodies.
- And the foremost step is Antibody Staining. I will be discussing the antibody staining in detail in subsequent chapters!
While every laboratory continues to optimize flow cytometry protocol, it traditionally involves the following steps:
- Cells are made a fix with formaldehyde to immobilize the desired proteins and their temporary signaling events.
- In test tubes, detergent or methanol is added in order to make the cells permeable to antibodies which in turn can get into their intracellular spaces.
- The optimal targetting of epitopes and antigens must be done by adding antibodies which are fluorescently tagged. And this can be done on multiple staining surfaces and intracellular proteins side by side.
- Now we are required to place the test tube in the flow cytometer and the fluid is then allowed to reach and then exit through the flow chamber, one cell at a time.
- As the cell crosses the beam of the laser, the light that bounces off is transmitted to light detectors.
- The data hence obtained from the experiment can finally undergo multiplex analysis to solve the unique aspects of the cells themselves and the trends being followed by them in cell signaling.
The staining of the antibody may differ accordingly.
- The direct staining methods have cells incubated in them with an antibody that is directly attached to a fluorochrome (e.g., FITC).
- In indirect staining, the fluorochrome-labeled secondary antibody is detected by a primary antibody that is not labeled. This method means that unattached primary antibodies can be lifted against many different targets, which enhances the choice of potent proteins for the researcher.
- In Intracellular, the staining takes place for intracellular antigens. The cellular proteins are traced back and tagged using a Golgi Block which is secreted by a cell afterward intracellular staining is done.
In a flow cytometry experiment, every cell passing through the flow cytometer is identified to classify as a definite incident. There is a type of representation for the Flow cytometry data which is typically depicted in one of two ways: one through histograms, which is used to measure or compare a single parameter, and another through dot-plots which compares 2 or 3 parameters concurrently on a two-or three-dimensional scatter-plot.
- Flow cytometry is used in pathology.
- The popularity of flow cytometry has gained momentum from its application in clinical pathology as well in oncology and prenatal diagnostics.
- We can use Flow cytometry in cell tracking, transduction, and phenotyping.
- Flow cytometry has a prominent use in cell sorting.
- We can segregate the dead and live bacteria with the help of certain dyes.
- Transfection efficiency can be determined when a fluorescent marker is used and hence we use flow cytometry.
- Flow cytometry can also be used in the detection and measurement of protein expression and post-translational modifications.
- Flow cytometry has potential use in cell proliferation assays.
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