Immunophenotyping

Immunophenotyping is a most important and regularly used technique in Immunology and Immunological research studies. The technique is usually used for the disease monitoring and diagnostic purpose either by immunohistochemistry or by utilizing the high-throughput technique Flow Cytometry.. Flow Cytometry is a common method for the immuno phenotyping the different cell subsets or sub populations of cells.

Immunophenotyping is used for the identification and characterisation of the cell surface markers and intracellular markers. In simple terms, the technique is used to detect and study the proteins expressed by the cells. Samples used for the study could be Tissue Section (Fresh or Fixed) or Cell suspension (mono-dispersed cell suspension for FCM). This technique helps in rapid and easy phenotyping of each cell in a heterogeneous sample according to the presence or absence of a protein combination. Markers (or proteins of interest) are usually functional membrane proteins which are involved in cell communication or signalling, cell adhesion or metabolism.

Fluorescent-conjugated antibodies are commonly used for the detection of the surface or intracellular proteins. (Fluorescent molecule is conjugated to an antibody, the antibody which is specific to a specific antigen present on the cells). Usually CD markers (Cluster of differentiation) are stained and investigated.

Main applications of this technique are, The detection and measurement of specific immune cell populations and subpopulations, Comparing the cancer cells to normal cells of the immune system.

Used for:

1) Lineage Assignment

2) Maturational characterization of malignant cells

3) Detection of clonality

4) Heterogeneity and aberrant features of the malignant cells

5) Quantification of Haematopoietic cells.

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Statistics in Flow Cytometry Data and "MFI" values

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Fluorescence Spectrum Viewers Tool.

Spectra Viewers are the fluorochrome tools which help in understanding the wavelength ranges of excitation and emission of fluorochromes.. These tools are very helpful in determining specific laser, detectors and range of fluorescence spectra. When Multi-Color Flow Cytometry is the experiment, then these must be used to analyze emission spectrum, fluorescence spill over and compensation values. How these tools help.. First, Lets consider a simple example of analyzing the spectrum for Green Fluorescence Protein (GFP) and you want to know the excitation wavelength and emission wavelength range for GFP. By knowledge, you know that GFP will excite with 488nm Blue laser and emits the green fluorescence at apporximately 510nm.. Now, just open a spectrum viewer and select the GFP as shown below. Now, you have analyzed GFP fluorescence and received the knowledge that It excites with 488nm Blue Laser and emission maximum is...

Compensation in Flow Cytometry (Fluorescence Spill Over Correction).

The term "Compensation" in flow cytometers refers to the process of correcting fluorescence spill over. The compensation matrix ensures that the fluorescence emitted is detected in a particular detector which is from the fluorescence being measured. As we all know that most of the fluorescence molecules have broad emission range. if more than one one fluorescence is selected, their emission spectra may overlap. To correct this spectral overlap in cytometry data, a mathematical matrix called compensation is used. The Intention of this article is to teach you the compensation from basic theory to practical. This example for FITC and PE fluorescence molecules shows that, Some of the FITC fluorescence is being detected in PE Detector. and Some of the PE fluorescence is being detected in FITC Detectors. Task here is to make sure that in cytometry data, FITC is in FITC detector only and PE is in PE Detector only....

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