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Understanding Flow Cytometry in a Simple Way!
#1
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.
[Image: flow_1.jpg]

How does it look like? What are it’s components?
Well, a typical flow cytometer looks like this:
[Image: flow2.png]

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: flow_3.jpg]
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):
[Image: flow4.png]
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
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


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#2
During the past ten decades the utilization of flow cytometry in the clinical labs has increased substantially. This is due to the development of less expensive, user friendly smaller instruments and also advancement in various clinical applications.

Immunophenotyping – It is the most frequently utilized application that is performed on the flow cytometer. This technique is used to identify and quantify heterogeneous populations of cells normally blood, lymph or bone marrow. In clinical labs it is useful in the diagnosis of leukemia and lymphomas (haematological malignancies).

Cell Sorting - The cell sorter is a flow cytometer that is specialized with the ability to isolate cells of interest by physical means into different collection tubes. This cytometer characterizes and interrogates every cell as it passes through a laser. The sorter then utilizes sophisticated and advanced electronics and fluidics to recognise and kick the cells of interest from the fluid stream into a test tube.

Cell Cycle Analysis – The four phases of the cell cycle can be analysed by Flow cytometry utilizing the fluorescent dyes. In addition to the determination of phases of cell cycle, this flow cytometry assay can also measure aneuploidy in the cells linked with chromosomal abnormalities.

Apoptosis – Also called as programmed cell death, is a usual part of the eukaryotic cell cycle. The two distinct forms of cell death that is apoptosis and necrosis can be differentiated and distinguished by flow cytometry by means of differences in biochemical, morphological and molecular events that occur in the dying cells.

Cell Proliferation Assays - Cell proliferation assays are extensively utilized in cell biology to determine metabolic activity of the cell in response to stimuli such as cytokines, growth factors and other components of the media. The flow cytometry is capable of measuring the cell proliferation by labeling the resting cells with a fluorescent dye of cell membrane, CFSE (carboxyfluorescein succinimidyl ester).

Intracellular Calcium Flux – By means of signal transduction pathways, the cells interact with their environment and also with each other. The flow cytometer can monitor the of calcium flux into the cell and quantify the extent to which cells are responding to the stimuli.

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#3
Hello Sunil Ji,
I am a fan of you, I never miss your threads and your posts in biotechstudents.com. I am a project trainee at RGCB, sorting cancer cells is a part of my project. This thread really helped me to understand Flow cytometry in a simple way. Thanks.
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#4
(08-28-2014, 03:59 PM)mario Wrote: Hello Sunil Ji,
I am a fan of you, I never miss your threads and your posts in biotechstudents.com. I am a project trainee at RGCB, sorting cancer cells is a part of my project. This thread really helped me to understand Flow cytometry in a simple way. Thanks.

That's really overwhelming. Thanks for the appreciation.
I'm happy to know that the post(s) helped you in some way.

All the best!
Cheers
Sunil
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


Suggested Reads:
Top Biotech Companies | Top places to work
Indian Biotech Companies and Job Openings
Aiming a PhD in Top Grad School? | These are the Important Points to Consider
Careers in Biotechnology | A list of various Options
Biotechnology Competitive Exams in India
Like Post Reply
#5
Flow cytometry really offers a simple yet a very useful technique in different fields of science including biotechnology. To gather thorough knowledge about various bio-processes, several analytical tools and techniques have been adopted. The data acquired not only helped in understanding different bio - processes, but also assisted in optimizing them to further develop new strategies of monitoring. Flow cytometry presents the option for precise and in depth analysis of cell populations.
Flow cytometry is now a universal method for rapid analysis of large numbers of cells individually with the help of measuring the scattering of light absorbance and fluorescence. Here every single cell passes through a laser beam along a directed stream of fluid. The interaction of each cell with the laser beam (i.e. Absorption, fluorescence or scattering) is monitored and the data, thus generated is then correlated with different characteristics of cell components. So through Flow cytometry scattered data of a cell population can be gathered without difficulty. The effectiveness of this technique lies in the facts that it can analyze a wide range of cellular parameters and at the same time it has the capability to attain information on the distribution of these parameters in the cell population. It was first used in medical sciences during cancer and chromosomal anomaly diagnosis. But soon it became a vital tool in the field of microbiology, biotechnology, pharmacology, toxicology and monitoring several bio-processes.
The recent growth in both instrument designing and software development with modern data acquisition system make possible in producing a range of specific staining assays thus making this tool even more useful.
Flow cytometric assays have been prepared to determine a variety of cellular characteristics. These include intracellular pH, membrane potential, size, etc. At the same time it has the potential to estimate the levels of cellular components such as surface receptors, protein, DNA and calcium. No doubt these measurements that give a clear picture of the distribution of these parameters in cell populations, are of immense importance in biotechnology. This information enables us better understanding of the population than that can be achieved through any other conventional methods used previously. There are even a few imminent challenges and limitations of the method which are demanded to be addressed through extensive scientific research and technology development and should be the future outlook. Hope this mini review on Flow cytometry gives the readers some valuable insight on flow cytometry and an enjoyable read.
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