Flow CytometryName: Md. Fayezur Rahaman.

Roll No:16VPATHJJ04MReg No: 37164

Department of PathologyBangladesh Agricultural University,

Mymensingh

TOPICS:

oDefinitionoprimary systems of the flow cytometeroPrinciples and data analysisoApplications

Flow Cytometry: Definition

Flow ~ in motionCyto ~ cell Metry ~ measure Measuring properties of single cells in a fluid

sample. Gives us the ability to analyze many properties of

many cells in very little time

In biotechnology, flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection by suspending cells in a stream of fluid and passing them by an electronic detection apparatus.

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 complexity

Monocytes which are medium sized Cell with slightly more

internal complexity

Neutrophils 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

ImmunophenotypingCell subsets are measured by labeling

population-specific proteins with a fluorescent tag on the cell surface. In clinical labs, immunophenotyping is useful in diagnosing hematological

malignancies such as lymphomas and leukemia.

DNA Content AnalysisThe measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as

propidium iodide, that intercalate into the DNA helical structure. The fluorescent signal is

directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses

in DNA.

Cell Cycle AnalysisFlow cytometry can analyze

replication states using fluorescent dyes to measure

the four distinct phases of the cell cycle. Along with determining cell cycle

replication states, the assay can measure cell aneuploidy

associated with chromosomal abnormalities.

ApoptosisThe two distinct types of cell

death, apoptosis and necrosis, can be distinguished by flow

cytometry on the basis of differences in morphological, biochemical and molecular

changes occurring in the dying cells.

Cell Proliferation Assays

The flow cytometer can measure proliferation by labeling resting cells

with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl

ester (CFSE). When the cells are activated, they begin to proliferate and undergo mitosis. As the cells divide, half of the original dye is

passed on to each daughter cell. By measuring the reduction of the

fluorescence signal, researchers can calculate cellular activation and

proliferation.

References

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