The Immunology Programme Flow Cytometry Lab in the Center for Life Sciences has 3 analysers and one cell sorter. The analysers are a Becton Dickinson FACSCalibur (2 lasers, 4 colours), Becton Dickinson LSR Fortessa (4 lasers, 15 colours), and a Beckman Coulter CyAn (3 lasers, 9 colours). The cell sorter consist of a BSL-2 pathogen compatible Sony Sy-3200 (4 lasers, 14 colors), and a Beckman Coulter Mo-Flo XDP (3 lasers, 8 colours). Commonly used fluorchromes which can be used on these systems include FITC, GFP, CFSE, PE, PI, APC, APC-Cy7, Pacific Blue, and the new Brillant Violet and Ultra Violet dyes from Becton Dickinson. The Flow Cytometry Lab also has a standalone PC with FlowJo software for analysis of flow cytometric data.
Seeing is believing! In 2015, the Department purchased the revolutionary ImageStreamRX Mark II (ISX) Imaging Flow Cytometer. This sate-of-the-art equipment combines the speed, sensitivity and phenotyping abilities of flow cytometry with the detailed imagery and functional insignts of microscopy. This unique combination enables a broad range of applications that would not be possible using either technique alone. The ISX houses 5 lasers (375 nm uv excitation laser, 488 nm blue excitation laser, 561 green excitation laser, 642 nm red excitation laser and a 785 nm darkfield laser), and features a 96-well atuosampler and a multi-magnification module (20x, 40x, 60x). The extended depth of field (EDF) feature dramatically extends the depth of field, enabling new applications and improving the performance of existing applications - especially spot counting applications such as FISH, autophagy and nuclear translocation.
The Operetta high content imaging platform delivers fully automated image acquisition and autofocus for reliable high content imaging from slides to multiple plates. The wide range of filters ranging fom Excitation: 360-400, 460-490, 520-550, 620-640 to Emission: 410-480, 500-550, 560-630, 650-760 which caters to a wide spectrum of fluorescent stains. Its transmitted light and digital phase contrast coupled with low to high magnification objectives of 2x, 20x, 40x, 60x, 100x and wide-field view allow better imaging option within the sample well. Its live cell chamber can maintain the correct environment conditions such as carbon dioxide level for keeping cell samples intact, and its kinetic analysis capabilities let you track those cells over time, quantifying dynamic behaviour such as chemokinesis, chemotaxis, and signal oscillations.
Being integrated to Operetta, the plate handler consists of a robotic arm and three racks with a total holding capacity of 42 plates. It provides high throughput automated imaging without any supervision over long duration of imaging. Moreover, the plate handler has a barcode reader that enables systematic tracking of individual plates and their images.
With Harmony High Content Imaging and Analysis Software, it allows assay development, genome-wide siRNA, compound screens using primary cells, stem cells, live or fixed cells. The analysis assays include: apoptosis, cell cycle, cell proliferation, cytoskeletal rearrangement, cytotoxicity, neurite outgrowth, protein expression, receptor translocation, signalling pathways.
The microscopic analysis of cells is becoming an increasingly important component of cell biology and immunology research. The Department bio-imaging room houses a variety of microscopes for research use. Depending on the application, users may choose between conventional light, inverted or fluorescent microscopy. Image capture and analysis functions are also available. For high-end bio-imaging need, researchers may also approach the Confocal Unit at NUMI (National University Medical Institute) or the neighbouring School of Medicine Electron Microscopy Unit where electron, confocal and atomic force microscopy services are available. The Advanced Imaging Laboratory in the Centre for Life Sciences has 2 confocal microscopes for Departmental use; a Leica SP5 inverted (405 nm, 488 nm, 543 nm, 633 nm laser lines) and a Zeiss LSM 510 upright (405 nm, 458/477/488/514 nm, 543 nm, 633 nm laser lines). This laboratory also boasts a Compucyte iCys Laser Scanning Cytometer for high-contnet image capture and analysis.
Traditional PCR is performed in a tube and when the reaction is completed, the amplified DNA fragments are analyzed and visualized by running an agarose gel. Real-time PCR (RT-PCR), however, allows quantitation of the starting amount of DNA, cDNA and RNA. In addition, RT-PCR can be used as a rapid assay as it is not necessary to subsequently perform a gel electrophoresis. Within the Department, there are two Applied Biosystem 7500 RT-PCR machines for common use which can accommodate up to 96 samples at a time using a microtiter plate format.
The ChemiDoc MP system is a full-feature instrument for gel or western blot imaging.It is designed to address multiplx fluorescent western blotting, chemiluminescence detection and general gel documentation applications. Its features are based on CCD high-resolution, high sensitivity detection technology and modular options to accomodate a wide range of samples and support multiple detection methods. The system is controlled by Image Lab software to optimise performance for fast, integrated and automated image capture and analysis of various samples.
The OminiLog Incubator-Reader uses Phenotype MicroArrays Technology to characterise cells for rapid identification of aerobic Gram-negative and Gram-postitive bacteria by producing a metabolic pattern from test reactions performed within a 90 well Biolog Microplate. This microplate is coated with 71 carbon source and 23 chemical sensitivity assays. After inoculation with a cell suspension, the plates are placed in the OmniLog incubator-reader, typically for 24 to 48 hours and their phenotypic behaviour recorded by a CCD camera. The unique metabolic pattern generated by the organism is compared to hundreds of identification profiles in a corresponding Biolog Database for analysis and organism identification.