The Flow Cytometry Facility has a four laser MoFlo™ MLS Flow Cytometer from Dako, Inc., operated exclusively by trained facility personnel. The four lasers provide excitations from 350nm to 752.5nm. This instrument has 10 photomultiplier tubes (PMTs) for collecting fluorescence data, plus a forward scatter detector for cell size/integrity measurements. Peak height, integrated area and pulse width data can be collected from each of our PMTs. A digital signal processor (DSP) computed-parameter board allows for up to 32 parameters (including pulse width on the trigger event and time) to be stored in list mode for a sample. In addition to analysis, samples can be isolated, based on combinations of the fluorescence and light scatter parameters, thus providing the investigator with a highly enriched population for subsequent analysis or culture. Sorting can be performed in bulk or in specific numbers of cells deposited into individual wells of multi-well plates.
Two analytical cytometers are also available for our users, the CyAn™ ADP from Dako, Inc. and FACScalibur™ from Becton Dickinson. The CyAn™ ADP has three excitation lines (405nm, 488nm, and 635nm) and nine fluorescent detectors. The FACScalibur™ has excitation capabilities of 488nm and 635nm and four fluorescent detectors.
The Laser Scanning Cytometry Facility has a Laser Scanning Cytometer, the iCys®, which is manufactured by CompuCyte. It has three excitation sources: 488nm, 635nm, and 405nm and eight possible filter sets. This instrument scans microscope slides and multiwell plates, saving fluorescence and/or transmitted light images. Cell contours can be generated, typically from DNA counterstain images, and flow cytometry type histogram and scatterplots can be used to classify cells into subsets.
The Molecular Imaging Facility consists of two types of instruments: the Typhoon™ 9410, manufactured by Amersham Biosciences, and an Odyssey® Infrared Imager from LI-COR. There are two Typhoon™ 9410 scanners available. They are used to scan phosphor screens, which have been exposed to radioactive sources on gels, blots, dishes, or trays. The phosphor screens are used to detect Beta and Gamma ionizing radiation from most isotopes. This allows linear quantification of data (over five orders of magnitude) and produces digital images, which are directly manipulatable for analysis and publication. Also, the Typhoon™ 9410 can be used to detect fluorescent labels on gels, blots, dishes, or trays. The Typhoon™ 9410 has four excitation sources, 457nm, 488nm, 532nm, and 633nm, which allow for use of a wide range of fluorescent dyes. There are also selectable emission filters (520BP40, 555BP20, 580BP30, 610BP30, 670BP30, 526SP, and 560LP), which allow for the collection of specific and multiple signals.
The Odyssey® Infrared Imager can be used to analyze two-color western blots, two-color in-gel westerns, two-color northern blots, and any other primary antibody tagged samples, which can be stained with near-infrared secondary antibodies (IRDye 700 and IRDye 800) or Alexa Fluor 680. It can also visualize Coomasie stained protein gels, which are fluorescent in the infrared. Along with scanning gels and membranes, it can also scan microplate cell-based assays. Various resolutions can be used for scanning, which range from 21u to 337u. This methodology allows for linear quantification of data (over three to four orders of magnitude) and produces digital images, which are directly manipulatable for analysis and publication.