Instruments at the Flow Cytometry Core Facility
BD Accuri (488nm and 633 nmlaser)
FACS DiVa Vantage Cellsorter (488nm, 633nm & UV line lasers)
Luminex’s Multianalyte Profiling (xMAP) System
488 nm and 640nm (rated at 20,000-h life)
Light Scatter Detection
Forward (0°, ±13°), Side (90°, ±13°)
• FL1 533/30 nm (eg, FITC/GFP)
• FL2 585/40 nm (eg, PE/PI)
• FL3 > 670 nm (eg, PerCP, PerCP-Cy5.5, PE-Cy™7)
• FL4 675/25 nm (eg, APC)
Flow cytometric analysis
Flow Cytometry is a process in which physical and/or chemical characteristics of single cells (or organelles / particles) are measured. The measurements are made as the cells pass through the measuring apparatus, a flow cytometer, in a fluid stream. The ability of flow cytometers to evaluate cells at an extremely rapid rate (e.g. up to 20,000 events per second) makes this technology ideally suited for reliable and accurate quantitative analysis of selected physical properties of cells of interest. Cells are stained with fluorescent probes which bind to specific cell associated molecules and allows measurements of various phenotypic, biochemical and molecular characteristics of individual cells (or particles) suspended in a fluid stream. As the cells flow past a focused laser beam of appropriate wavelength, the probes fluorescence and the emitted light is collected and directed to appropriate detectors (optics). These detectors, in turn, translate these light signals into electronic signals proportional to the amount of light collected. Information regarding the relative size and granularity of a cell, for example, is also obtained as these characteristics influence the way in which light is scattered as the cell passes through the laser beam. The sensitivity of these instruments for detecting the presence of molecules expressed at low levels is impressive; given high quality cell preparations and reagents, as few as 50 molecules per cell may be detected.
FACS DiVa Vantage Cell Sorter
Laser Excitation• 488nm, 633nm & UV line lasers
Emission Detection• 8 parameters (2 scatter & 6 fluorescence)
• Sort four populations simultaneously in tubes
• Sort into 6, 24, 48 and 96 well plate or onto slides
• High pressure sort (45psi) with up to 10 000 cells sort/sec
• Sort onto slides
• Sterile sort
• Cooling of sample and collections tubes (4oC- RT)
• Advanced analytical software (BD FACS DiVa Software) for plots and statistics
A cell sorter is a flow cytometer that is able to select target cells and collect these separately from the rest of the cell suspension. Cell sorters have the ability to electronically deflect cells (sort) with defined properties into separate collection tubes, plates or slides. For cell purification, flow cytometry is especially well suited for applications requiring high purity (up to 99.5 %). The purity of sorted cells may be validated by rerunning the sorted fraction and purity data is easily determined by statistical software analysis. Cellsuspensions stained with multiple fluorochromes (e.g. up to six distinct fluorescent probes reacting with different cellular properties) may easily separate complex mixtures of cells, based on multiple marker expression.
Luminex IS 100
|Luminex IS Multianalyte Profiling (xMAP) System
• Bio-Plex Luminex xMAP machine
• computer workstation
• vacuum manifold
• orbital shaker
• the workstation has all the required basic laboratory articles required for
Luminex IS Multianalyte Profiling (xMAP) is an easy-to-use protein analysis system that permits the simultaneous quantitative analysis of up to 100 different proteins, peptides, or DNA molecules in a single microtiter well. The 96 well plate run allows you to assay up to 80 analytes/plate (sample size 25 uL). This multiplexing feature allows to obtain massive information, using a minimum amount of sample. Multiplexing enables a comprehensive overview of complex biological systems in either serum, plasma, cell supernatant or tissue, spinal fluids, urine or extracts of either cells or nuclei . In addition, sample volume requirements are reduced and assay throughput is dramatically increased.
Potential applications include:
• functional genomics
• validation of drug leads
• secondary screening
• as well as high-throughput ELISA and Western blot assays
Elutriation -high troughput cell separation
The Flow Cytometry and Cell separation Core Facility has a platform that offers centrifugal elutriation for isolation of monocyte-enriched fractions of peripheral blood mononuclear cells and separation and purification of peripheral blood Lymphocytes.
Mononuclear cells (MNC) are isolated by initial ficoll gradient treatment, prior to elutriation. The MNCs are loaded unto the elutriation chamber, where cells of different sizes are balanced by centrifugal force and counterflow. By balancing centrifugal force against the opposing buffer flow, lymphocytes, which are about 6-8 µm and monocytes, which are 8-10 µm, will be selectively removed from the mixture. From one elutriation run, it is possible to harvest between 50-100 million cells with a purity ranging from 90-97%.