Fluorescence Cytometry Core

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What is Flow Cytometry?
What is LSC?

Go With the Flow...
What is Flow Cytometry?

Flow Cytometry, sometimes referred to as FACS™ (Fluorescence Activated Cell Sorting), is a very useful technology to accurately measure many biological or chemical properties of cells or events.  Individual cells held in a thin stream of fluid are passed through one or more beams of highly focused laser light.  Emitted light from fluorochromes attached to surface or intracellular markers are detected and transformed to data.  The technology is powerful because as individual cells are analyzed, small subpopulations can be identified, and small differences in expression of cell markers are distinguishable.  Another added benefit is the relatively small amount of sample needed to identify multiple parameters.  Because the FACSAria cytometer has three different wavelengths of lasers and nine optical filter pathways, an enormous amount of data can be generated from each cell.  Up to nine different identifying markers, as well as two parameters of scattered light can categorize each cell.  This aids in understanding the unique bio-chemical processes that occur on the cellular level.

Once identified, cells can then be sorted with very high purity into as many as four different vessels.  An electric charge is applied to the stream which has been shaken or vibrated to break up into individual droplets.  The droplets containing the desired cells are then deflected into collection tubes.  Cells can be sorted at speeds of up to 25,000 cells/second.  Single cell sorting into plates with wells is possible as well for cloning.  These purified collections can then be further analyzed or used for a variety of ongoing investigations.

APPLICATIONS:

Immunophenotyping:  Immunophenotyping involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin.  This method is often used to label molecules on the cell surface, but antibodies can be directed at targets within cytoplasm.

DNA Staining:  FACS is used to study DNA content.  Propidium iodide (PI) and Hoechst dyes bind to DNA and become fluorescent.  DNA staining can be used to study the cell division cycle.  Relative DNA content shows the proportion of cells in G1, G2 and S phases.

 

Apoptosis Studies:  Several different indicators of apoptosis are commercially available with fluorescent indicators making Flow Cytometry an ideal tool for measuring cell death.  It is possible to quantitate live, apoptotic and dead cells in a variety of treated conditions.

 

Calcium Flux and Other Metabolic Studies:  Metabolic characteristics such as calcium flux, mitochondrial activity, pH, and free radical production can be measured in live cell populations in real time.

 

Gene Expression and Transfection:  Flow Cytometry is used to measure gene expression in cells transfected with recombinant DNA incorporating fluourescent proteins (GFP,etc.)  Transfection efficiency can be determined and sorting of transfected cells can be done for purification for further use.

Plan to Scan…
What is Laser Scanning Cytometry?

Laser Scanning Technology combines the technologies of Flow Cytometry, Image Analysis and Automated Fluorescent Microscopy.  The Compucyte LSC™ uses three lasers (Argon, HeNe, & U-V diode) and four detectors (PMTs) to visualize and quantify fluorescence of many dyes and probes on microscope slide specimens.  A solidstate sensor measures forward scatter light.  The automated motorized stage can scan thousands of cells in minutes giving a high degree of statistical power over manual methods.  Data is displayed in real time.  CompuSort™ relocates gated cells and creates cell galleries in seconds.  Color images are obtained by a monitored CCD video camera and a Kodak™ DC120 digital camera.

Representative of FISH studies

 

Representative data and images of nuclear and cytoplasmic staining