Flow Cytometry technique

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Flow Cytometry Name: Md. Fayezur Rahaman. Roll No:16VPATHJJ04M Reg No: 37164 Department of Pathology Bangladesh Agricultural University, Mymensingh

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Flow Cytometry

Name: Md. Fayezur Rahaman.Roll No:16VPATHJJ04MReg No: 37164Department of PathologyBangladesh Agricultural University, Mymensingh

TOPICS:

Definitionprimary systems of the flow cytometerPrinciples and data analysisApplications

Flow Cytometry: Definition

Flow ~ in motionCyto ~ cell Metry ~ measureMeasuring properties of single cells in a fluid sample.Gives us the ability to analyze many properties of many cells in very little time

Inbiotechnology,flow cytometryis alaser-based, biophysical technology employed incell counting,cell sorting,biomarkerdetection by suspending cellsin a stream of fluid and passing them by an electronic detection apparatus.

Fluorescence-activated cell sorting (FACS)

Cell sorting is the ability to separate cells according to their properties. These properties can be described as intracellular (inside the cell) or extracellular (outside the cell). Intracellular processes can include DNA, RNA and protein molecule interaction, whereas extracellular physical properties include size, shape (morphology), and surface protein expression.

A biomarker is a measurable indicator of the severity or presence of some disease state. For example, rubidium chloride is used in isotopic labeling to evaluate perfusion of heart muscle. More specifically, a biomarker indicates a change in expression or state of a protein that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment.

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A Flow Cytometer

Place your sample here

Lasers: Which are the light source for scatter and fluorescence.The optics : Which gather and direct the light.The detectors: Which receive the light.

The electronics and computer system: Which convert the signals from the detectors into digital data and perform analysis

Fluidic system: which presents sample to the interogation point.This view shows the primary systems of the flow cytometer.

WORKING PRINCIPLES AND DATA ANALYSIS

Flow cytometry performes analyses by passing thousands of cells per second through a laser bean and capturing the light emerges from each cell passes through.

The interrogation point:It is the heart of the system, where the laser and sample intersect and the optics collect the resulting scatter and the fluorescense.

For accurate data collection it is important to particles/cells are passed through the laser beam one at a time. How the sample pass through the interrogation point?

The scattered light received by the detector and transmitted into a voltage pulse.Small cell produce small scatter and large cell produce large scatter . The magnitude of the voltage pulse recorded for each cell is proportional to the cell size.How laser light is used to detect individual cell in the stream?Forward scatter:

If we plot a histogram of these data small cell appears to the right and larger cell appear to the left

Side scatter:

These side scattered lite is focused through a lense system& is collected by separate detector usually located 90 from the laser path.

When create a scatter plot using forward and side scatter data, lymphocytes which are small cells with low internal complexityMonocytes which are medium sized Cell with slightly more internal complexityNeutrophils and other granulocytes which are large cell, that have a lot of internal complexity

Finally when laser light of right wave length strikes the flurophore a fluorescence signal is originated and detected by the flow cytometer.

One of the most common ways to study cellular character using flow cytometry by using fluorescence molecule such as fluorophore labeled antibodies.

In this experiment the labeled antibody added to the cell sample. The Ab then binds to specific molecule on the cell surface or inside the cell.

Fluorescence Detection

The fluorescent light coming from labeled cells as they pass through the laser travels along the same path as the side scatter signal. As the light travels along this path it is directed through a series of filters and mirrors so the particular wave length ranges are determined to the appropriate detectors.

One color histogram:

Fluorescence data is collected In generally the same way as forward and side scatter data.The fluorescent light is then detected to the appropriate detector where it is translated into a voltage pulse proportion to the amount of fluorescence. All of the voltage pulse are recorded and graphically presented.

Two color dot plot:If we analyze data from the 2 color experiment using a scatter plot 4 distinct population will be found.

Cells with bright orange fluorescence appear in the upper left quardent.Cells with green fluorescence appear in lower right quardant.Cells with both bright green and bright orange appear in upper right quardent.Cells with both low green and low orange appear in lower left quardent.

Applications

Referenceshttp://flowcytometry.berkeley.edu/pdfs/Basic%20Flow%20Cytometry.pdfhttp://www.azom.com/article.aspx?ArticleID=6020https://www.beckmancoulter.com/wsrportal/wsr/industrial/particle-technologies/coulter-principle/index.htmhttp://www.cyto.purdue.edu/cdroms/cyto2/6/coulter/ss000103.htmhttp://ajcp.ascpjournals.org/content/134/2/271.full.pdf+htmlhttp://cancerres.aacrjournals.org/content/43/9/3982.full.pdf+htmlAppl.%20Environ.%20Microbiol.-1990-Amann-1919-25.pdfhttp://europepmc.org/abstract/med/2645625http://www.clinchem.org/content/46/8/1221.fullhttp://www.icms.qmul.ac.uk/flowcytometry/uses/cellcycleanalysis/cellcycle/index.htmlKuby Immunology, 7th Editionhttp://www.clinchem.org/content/46/8/1221.fullhttp://www.seattlechildrens.org/research/cores/flow-cytometry/applications-of-flow-cytometryhttp://www.d.umn.edu/~biomed/flowcytometry/introflowcytometry.pdf