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Sankara Nethralaya
Sankara Nethralaya
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Central Research Instrumentation Facility
BD FACS CALIBUR
Faculty incharge-Dr.K.Lily Therese

INTRODUCTION:

The technique of analyzing individual cells in a fluidic channel was first described by Wallace Coulter in the 1950s, and applied to auto¬mated blood cell counting. Subsequent developments in the fields of computer science, laser technology, monoclonal antibody production, cytochemistry, and fluorochrome chemistry led to the development of the flow cytometer two decades later. The present “state-of-the art” flow cytometers are capable of analyzing up to 13 parameters (forward scatter, side scatter, 11 colors of immuno¬fluorescence) per cell at rates up to 100,000 cells per second..

BD FACS CALIBUR:

The FACSCalibur system is a modular, bench top flow cytometer from Becton Dickinson Immunocytometry System (BDIS). It has been designed for applications that range from routine clinical to advanced research, analyzing single cells as they pass through a focused laser beam. It is a tricolor, automated system that is capable of analyzing a wide variety of reagents.
The FACSCalibur technical specification capabilities include a 488nm, air-cooled, Argon-ion laser, excitation/emission optics, fluidics and signal processing.

To book Instruments Call : 28271616 (1325), 28272727

PRINCIPLE:

Prepared single cell or particle suspensions are necessary for flow cy¬tometric analysis. Various immunoflurescent dyes or antibodies can be attached to the antigen or protein of interest. The suspension of cells or particles is aspirated into a flow cell where, surrounded by a narrow fluid stream, they pass one at a time through a focused laser beam this process is termed as HYDRODYNAMIC FOCUSSING.

The light is either scattered or absorbed when it strikes a cell. Absorbed light of the appropriate wavelength may be re-emitted as fluorescence if the cell contains a naturally fluorescent substance or one or more fluorochrome-labeled antibodies are attached to surface or internal cell structures.

Light scatter is dependent on the internal structure of the cell and its size and shape. Fluorescent substances absorb light of an appropriate wavelength and reemit light of a dif¬ferent wavelength. Fluorescein isothiocyanate (FITC), Texas red, and phycoerythrin (PE) are the most common fluorescent dyes used in the biomedical sciences. Light and/or fluorescence scatter signals are de¬tected by a series of photodiodes and amplified. Optical filters are es¬sential to block unwanted light and permit light of the desired wave¬length to reach the photo detector. The resulting electrical pulses are digitized, and the data is stored, analyzed, and displayed through a computer system.7, 8 The end result is quantitative information about every cell analyzed (Fig. 1). Since large numbers of cells are analyzed in a short period of time (>1,000/sec), statistically valid information about cell populations is quickly obtained.

APPLICATIONS:

CLINICAL APPLICATIONS

Immunophenotyping Leukemia and Lymphomas and Monitoring Residual Disease
Determining CD34 counts for Hematopoietic Reconstitution
Monitoring Organ Transplant Patients for Rejection
Reticulocyte Counts and Diagnosis of Paroxymal Nocturnal Hemoglobinuria (PNH)
DNA analysis of S-phase fraction of solid tumors

RESEARCH APPLICATIONS

Antibodies can be used to identify cells and cytokines or cytokine receptors to identify specific populations of functional cells.
It is also possible to use fluorescent probes to many physiological functions such as metabolic processes, ion channels, organelles and intracellular pH.
These assays are extremely important in understanding the effects of drugs on cell physiology.
Finally, molecular phenotyping using in situ PCR, in situ hybridization and the use of fluorescent markers for isolation of transfected cells, such as green fluorescent protein, are significantly increasing.

FUTURE EXPECTATIONS:

The flow cytometer is a versatile tool with enormous potential for the study of cells and particles. Because of its unique analytic capabili¬ties, the flow cytometer has become an integral part of the medical research laboratory during the past two decades. Immunophenotypic analysis and lymphocyte subset analysis is widely performed in the clinical laboratory, but most of the other applications are limited to larger and/or specialized laboratories. However, no other laboratory instru¬ment provides multiparametric analysis at the single cell level, and the flow cytometer or application-variants of the flow cytometer will become more valuable as medical diagnosis and therapy changes.