Summer Research Fellowship Programme of India's Science Academies

Flow cytometer as a tool in understanding immune system

Hilal Ahmad Ganie

Central University of Punjab, Bathinda, 151001

Professor Amita Aggarwal

Sanjay Gandhi Postgraduate Institute Medical sciences, Lucknow, 226014


Both in research and in clinical practice of Medicine we often rely on the quantitative estimation of our target cell, protein or other analytes. This estimation can be done by multiple methods however in recent years flow cytometry has become a powerful tool not only to study cells but also to measure soluble products like antibodies, cytokines etc. Flow cytometer works on the principle of light scattering, excitation of fluorochromes tagged to particles and detection of light signals released by the excited fluorescent molecules. Our immune system is a complex system of cells and organs. The quantification of immune cells and molecules released by them needs sensitive instruments like flow cytometer, which can not only quantify different types of cells based on their surface markers but it can also help us in accessing their function as well. Using flow cytometer, we can perform the basic investigation of immune system like number and percentage of different immune cells, viability of cells etc. Similarly, intracellular molecules like reactive oxygen species in phagocytic cells can be detected. We can also detect the production of different cytokines produced by different cell lines. Such assays are often used in research to detect the production of different signaling molecules during different treatments or during different induced infections. We can also access the phagocytic activity of neutrophils and monocytes and a lot more. Actually, it is possible to assess multiple functions as well as phenotype of a single cell using flow cytometry. Due to its high sensitivity and specificity, flow cytometer has replaced techniques like ELISPOT.

Keeping in mind the importance of flow cytometer in research I choose to learn this technique, its principle, working, how to analyze results of flow cytometric data and how to design experiments for basic and clinical research. As I am a microbiology student, I have learnt techniques that are used to assess immune functions related to microbial infections. These experiments will help me in understanding the instrument and its importance in research.

Keywords: flow cytometer, flourochromes, fluorescence, antigen detection, cell viability, cell cycle.


My objective was to understand the principle working and use of flow cytometer in research field of immunology and microbiology. To understand all this i performed following experiments. The importance, principle, protocol and results of each experiment are discussed seperately.

FSC Forward scattering
SSC Side scattering
NKC Natural killer cell
ROS Reactive oxygen species
7AAD 7-amino actinomycin D
PI Propidium iodide
PBMC Peripheral blood mononuclear cells
PBS Phosphate buffered saline
CD Cluster of differentiation
FACS Fluorescent activated cell sorting
APC Allophycocyanin
FITC Fluorescein isothiocyanate
PE Phycoerythrin
DHR Dihydrorhodamine
PMA Phorbol myristate acetate
PAMP Pathogen associated molecular patterns
TLR Toll like receptors
ELISPOT Enzyme linked immunospot assay
LPS Lipopolysaccharide
BFA Brefeldin
PHA Phytohemagglutinin
IFN Interferon
BSA Bovine serum albumin
PNCA Proliferating nuclear cell antigen
Written, reviewed, revised, proofed and published with