The Flow and Image Cytometry Resource

The Leica TCS SP2 system was designed for high resolution confocal image acquisition using laser point scanning of living and fixed specimens. This system is ideal for fixed specimens that require severe background suppression and cross-talk correction as well as detecting far red fluorescence not detectable by eye. The system also has 3D reconstruction capabilities as well as simultaneous multi-spectral scanning and image acquisition.

Technical Specifications:

• TCS SP2 AOBS Spectral Confocal Scanner mounted on a Leica DM IRE2 inverted fluorescent microscope
• 4 lasers (diode, argon, and 2 helium neon)
• 4 prism spectrophotometer detectors
• 405, 458, 476, 488, 514, 543, and 633nm laser excitation lines
• Adjustable emissions bandwidth for customized collection of fluorescence
• 10X dry, 20X (dry, water, oil), 40X and 63X oil immersion objectives
• Motorized stage and Galvanometer z-stage for high speed and precision
• Transmitted light detector and manual condenser (allows overlay of transmitted light image with fluorescence image
• Leica Confocal Software (LCS) for multi-dimensional image series acquisition, image processing and analysis, 3D reconstruction and measurement, FRET, FRAP, Lambda Scanning, dye separation , multicolor restoration and controlling the motorized microscope stage

Applications:
Some useful applications of a confocal system include but not limited to: acquisition of high quality fluorescence images of a single optical slice, acquisition of images from thick section s requiring severe background suppression, reconstruction of 3-D images, co-localization and expression studies, kinetic studies (FRAP).

The Flow Sorting Facility houses two cell sorter instruments, FACSAria I and FACSAria II. The FACSAria I was upgraded in September 2010 and is equipped with 18 fluorescent PMT's and 405, 488, 561, and 640 nm lasers, and has bio-containment capabilities. The FACSAria II is equipped with four lasers (355 nm, 408 nm, 488 nm, and 633 nm) and has 13 fluorescent detectors. Both instruments have the capacity to do four-way sorts as well as sorting into microtiter plates (96-, 48-, 24-, 12-, or 6-wells) and onto slides.

Our facility maintains a CLIA-certified laboratory that provides processing and flow cytometry analysis services for diagnostic patient samples and clinical experimental protocols. The lab receives samples from all over the nation.

The Imagestream Platform:
The ImageStream platform (Amnis Corp.) is a novel flow-cytometric technology that is able to capture high-resolution images of cells in flow at rates of up to 1000 cells per second. By combining the high-throughput power of flow cytometry with its imaging capabilities, ImageStream cytometry allows for acquisition of statistically robust data on the detailed structure of cells and the location of intracellular components, effectively combining the strengths of microscopy and flow cytometry to achieve large scale detailed cell analysis.

Instruments:
ImageStream-100: 405 nm, 488nm, 561 nm Excitation Lasers; 6 channel detectors; 40x objective; Extended depth of field (EDF) option ImageStream-X: 405 nm, 488 nm, 561 nm, 658 nm Excitation Lasers; 12 channel detectors; 20x, 40x, 60x objectives; Extended depth of field (EDF); autosampler (96 well plate format)

Applications:
Current applications at RPCI include cell signaling studies (incl. NFκB, NFAT, STATs, beta-catenin), cell-cell interaction studies (e.g. antigen presenting cells with effector cells), intracellular trafficking of fluorescent probes or (co-)localization with cell organelles, fluorescent in situ hybridization.

Example: quantitative analysis of nuclear translocation of NFκB
The IDEAS® image analysis software applies features (algorithms) and masking
operations (region-finders), to perform image-based analysis. To assess events of
translocation, a similarity score algorithm is used. This is calculated by log
transforming the Pearsonʼs correlation coefficient for pixel values of the nuclear and
transcription factor images (eg DRAQ5 vs FITC-NFkB-p65). Changes in the
similarity score distributions are reported as an Rd metric, which is a measure for
the shift of two distributions (Fisherʼs Discriminant Ratio).
Similarity Score Rd Value

Luminex may be a misleading term. The actual assay refers to a bead based multiplexing technique which in large part was developed right here at RPCI under the leadership of Carleton Stewart. The Luminex Corp. is the leading company that has commercialized various applications of this technique.

The basis of the Luminex assay is the xMAP (any Multiple Analyte Profiling) technology which color-codes microspheres with different combinations of 10 shades of 2 dyes creating a 100 bead sets that can be discriminated by flow cytometry. Each bead can be coated with an analyte specific capture reagent (antibody) and binding of the reagent can then be quantified using reporter antibodies. Thus, xMAP technology allows multiplexing of up to 100 unique assays within a single sample. The Luminex-100 instrument has 2 lasers in which one is used to excite and detect a specific bead and the other to detect the PE bound to a reporter antibody.

In our facility, the assay is performed in a 96-well plate format of which commonly 80 wells are available for sample analysis (16 wells are used for internal standards and controls). Thus in principle, 100 analytes can be assayed on 80 samples in a single experiment.

Common applications: protein expression profiling (detection of cytokines, chemokines, phospho-proteins, cell signaling, isotyping, apoptosis, etc. in serum, plasma, cell culture supernatant, cell lysate). For a full repertoire of available analytes please see the websites of our preferred vendors. Links top these web sites are provided on the right.

Actual costs for the assay are dependent on a number of variables including the number of samples, the number and specific characteristics of the analytes you are interested in and the level of service requested. For a detailed estimate on your specific study, please contact us.

Links for Service Request Forms, accepted and preferred sample preparation, available services and associated rates are provided on the left.

The latest applications of the soluble bead array technology are molecular-based assays in which the beads are coated with nucleic-acid capture sequences rather than antibodies. Applications of this multiplexing approach are in the field of SNP detection and miRNA detection. If you have specific interest in these applications please see the vendors' web sites or contact the facility.

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