FLOW CYTOMETRY

BY- SAI MANOGNA (MSIWM014)

Flow Cytometry : Flow cytometry is an efficient technique, because it enables the physical and chemical properties of cells up to thousands of particles per second in a heterogenous population. Multiple parameters of single cells can be analyzed simultaneously using this approach. This makes it a quick and quantitative technique for the analysis and purification of suspended cells. We can decide the phenotype and work using flow cytometry and even sort live cells.

It is primarily used to measure the fluorescence intensity provided by the protein- detecting fluorescent labelled antibodies, or ligands that bind to specific cell associated molecules such as DNA-binding propidium iodide. The staining process involves creating a single cell suspension from samples of cell culture or tissue. Cells are then incubated and tested on the flow cytometer in tubes or microtiter plates with unlabelled or fluorochrome- labelled antibodies.

Principle : Flow cytometry is used when a large number of different cell types need to be profiled in a population. On the basis of variations in size and morphology, the cells are separated. In addition to recognizing and segregating different subpopulations, fluorescently-tagged antibodies targeting the particular antigens on the cell surface may be used.

Components in flow cytometer :

1. A flow cell
2. A measuring system
3. A detector
4. An amplification system
5. Computer analysis of the signals.

1. A flow cell : this has a liquid stream containing the cells so they pass only single cells through the beam for sensing.
2. Measuring system : This uses the measurement of conductivity and optical system lamps such as mercury and xenon, high power water cooled lasers such as argon and krypton, diode lasers such as blue, green. Red, violet resulting in light signals.
3. Detector : The detector converts the measurement of forward scattered light and side scattered light as well as dye specific fluorescence signals into digital signals which further can be processed by computer.

Flow Cytometric Analysis :

 A.  Functional analysis : This method can determine the biological activity of cells including reactive oxygen species production, changes to the mitochondrial membranes during apoptosis, rates of phagocytosis in labelled bacteria, indigenous calcium content and changes in metal content during drug reaction, etc.

B.  Cell cycle analysis : The amount of DNA present in each phase of cell cycle varies. The fluorescent dyes which bind to DNA or monoclonal antibodies which can detect antigen expression, can evaluate this variation in the content of DNA. This approach can also be used to quantify other factors including cell pigment content such as chlorophyll, DNA copy count variance, intracellular antigen, enzyme activity, oxidative bursts, glutathione and cell adherence.

C.  Apoptosis and Necrosis Assessment : Apoptosis or programmed cell death is followed by typical changes in morphology of cells, structure loss, cell detachment, cytoplasm condensation, cell shrinkage, cell residue phagocytosis and nuclear envelope changes. Oncosis is a necrotic occurence in which a cell tends to swell instead of to decrease in its size. The plasma membrane breaks down and proteolytic enzymes are released, which can also damage the surrounding tissue. These changes can be analysed by flow cytometry in plasma membrane and cell type.

D. Determination of cell viability : This approach can also be used to test cell viability following addition of pathogens or drugs. Any defects in the integrity of a cell membrane can be assessed with the usage of dyes that can penetrate the cell membrane. Fluorescent samples like bis-oxonol will bind to proteins on the cell membrane, so that different stages of necrosis are established.

Working of Flow cytometry :

During the flow cytometry, a sheath fluid concentrates the cell suspension hydrodynamically through a small nozzle, so that only one cell can pass the laser at a time. A detector is positioned in front of the laser beam to capture the forward scattered light from the cells, while multiple detectors are also mounted on to the sides to determine the amount and intensity of scattered light in each direction.

As of now we understood that there are two ways in which the light signals can be analysed;

They are:

1. Scattering
2. Fluorescence emission

1. Scattering :
   
   i. Forward scattering : forward scattering refers to the light refracted by a cell which is moving in the same direction as it was initially moving. The proportion of light scattered i.e, forward scattered determines the cell size, where larger particles emit more forward scattered light than the smaller particles, and also larger cells will have a stronger forward scatter signal.
   
   ii. Side scattering : Side scattering refers to the refracted light that is orthogonal to the light path direction. This provides information about granularity in which highly granular cells emit more light than cells with low granularity.
   
   For instance, cells with large granules, emit high forward scattered and high side scattered light. Monocytes that show low granularity emits high forward scattered light but low side scattered light. Therefore, based on the forward and side scattered light proportions, different types of populations can be differentiated.

Forward scattered light is directly proportional to cell size and refractive index.
Side scattered light depends on the shape and granularity of the cells.

1. Fluorescence emission :  apart from the forward and side scattered light, various cell types may also be segregated by fluorescent molecules. Fluorescent light may be emitted by fluorescent molecules after excitation by a compatible wavelength laser. Fluorescent light may originate from naturally occurring fluorescence materials in the cell such as NADPH and FAD; (this mechanism is termed as autofluorescence), or may originate from the fluorescent dyes or fluorescence-tagged antibodies that have been used to label a specific structure of the cell.

Data Analysis : Each cell passing through the ;laser light is detected as a separate event. A distinct channel is often assigned to various forms of scattered light i.e, forward-scattered, side-scattered and fluorescence emission wavelengths. The data is separately plotted for each of these occurrences and can be interpreted by two methods: Histograms and Dot-plots.

Histograms :

• Fast to read and easy to understand
• Most useful when only one parameter is important
• Representation includes intensity of single channel on x-axis and number of detected events on y-axis.
• Multiple overlaid histograms : used to compare single parameters from two different sample populations.
• 

Dot-plots :

• Most useful for multi-parametric data
• Can be 2-D or 3-D
• Each distinct event is represented as a single dot and intensity of each channel is represented on its own axis.
• More complex.

Uses of Flow cytometry:

1. Cell counting
2. Cell sorting
3. Determining cell characteristics and function
4. Detecting microorganisms
5. Protein engineering detection
6. Biomarker detection
7. Diagnosis of health disorders such as blood cancers