[SunilNagpal]

What is Flow Cytometry?
It's a 'Tool' that can isolate specific types of cells from a pool of cells. It is usually done by suspending cells in a stream of fluid and passing them by an electronic detection apparatus.


How does it look like? What are it’s components?
Well, a typical flow cytometer looks like this:


How does it Function?

It sorts the cells based upon their size, shape, color of light they fluoresce.
Once the cells are bombarded with energy, detector starts making following measurements:

1. Changes in Light Scattered (Decided by Shape and size)
2. Changes in Light Absorbed (Decided by Shape and Size)
3. Changes in Light Emitted (Decided by nature of cells and kind of fluorescent tag they are having)

Based on these measurements, specific types of cells are recognized and sorted by the sorter by manipulating their charge.

Image Source: Introduction to antibodies, 2nd Edition, Chemicon International, Page 28

HOW DOES THIS CELL SORTING COME TO USE?

Well, the applications are many, but let me explain two very basic and most frequent applications of Flow Cytometry:

1. POSITIVE SELECTION AND ISOLATION OF CLONES

Imagine a situation where you have inserted the gene of your interest in the target cell with a GFP reporter. Now, flow cytometry can come handy in this situation to select and isolate all the positive clones expressing the GFP and hence your gene of interest! And it does so extremely fast and precisely!
The practice is very common for positive selection of transfected mammalian cells.

2. DIAGNOSIS OF DISEASED STATE ( CANCER etc)

Now, Imagine another situation, where you want to diagnose a disease like cancer. All you need to do is take the sample of the patient’s body cells. Add a flourochrome labeled antibody to the sample, specific to the epitopes of the diseased state cells. The detector will detect the signal intensity of light emitted by the antibody labeled cells. Cell sorter will sort out the diseased state cells for further verification. So, this solves the dual purpose: intensity of signal gives the idea of condition of disease, and sorter gives the pool of diseased cells for further verification.

SOME DETAILS ON DETECTION?

All kinds of light signals (mentioned above), be it emitted/scattered/absorbed are transformed into digital signals using a computer program. The most common way of presenting the signals is through a single parameter graph of INTENSITY versus NUMBER OF CELLS (see figure below):

Courtesy: Introduction to Antibiotics by Chemicon

Such plots as depicted above indicate the fluorescence intensity detected against the number of times it was detected.

It is worth mentioning here that there are two kinds of flourochromes commonly used in Flow Cytometry:
1. Fluoroisothiocyanate (FITC)
2. Phycoerythrin (PE)
The reason they are the most preferred fluorescent tags is that both of these tags can be excited with the same laser wavelength i.e 488nm and both these have a well separated / distinguishable emission spectra i.e FITC having green spectra (530nm) and PE having orange emission spectra (570-575nm).

POSSIBILITY OF MULTIPLE KINDS OF DETECTIONS?

The best thing about Flow cytometry is that it can detect and sort multiple kinds of characteristics of a single type of cell. During multiple parametric detection, the signals are recorded in 2 dimensional or 3 dimensional diagrams, where in the intensity of one parameter’s response is plotted against the other (X vs Y plot i.e 2D) and cell is sorted based upon the response of combined intensity of the two parameters. So, if a population of cells is expressing two kinds of abnormal traits, then they can be labelled for both the characteristics and passed through the flow cytometer. It will detect the relative intensity of two signals from every cell and plot it as XY scatter/dot plot, giving an idea about the intensity of expression of the two traits in the cell in terms of which trait is expressed more and in how many cells.

Concluding

Flow Cytometry is thus a really simple yet very significant tool in scientific world, offering it’s services in wide range of applications like clinical research, disease diagnosis, routine scientific research (cloning, cDNA library preparation, toxicity profile monitoring, detection of pollutants and their effects etc). The ease of analysis and detection of the signals makes it a strong choice of both the naïve and experienced researchers. Hardly any submission of publication based on Flow Cytometry data goes unpublished, owing to the uniqueness and precision of the data generated by this high throughput tool! A basic understanding of this wonderful tool in Biotechnology is thus always expected of every biotechnologist, and I hope this article helped you in some way.

Thanks