Instrumentation

 
   FACSVantage with TurboSort: This flow cytometer is capable of high speed cell interrogation and sorting. The electronics of the instrument have recently been redesigned for pulse process speeds in excess of 2 MHz. This increased speed is reflected in viable cell recoveries in excess of 70% at sorting speeds of 20K/sec. This instrument has other modifications to provide advanced flow cytometry applications.

• Forward Scatter Detectors: These include both forward angle scatter and forward angle fluorescence detection (FFD). FFD is a new parameter that may be useful in providing additional cell resolving power.
• Seven color detection: Standard flow cytometry instrumentation provides up to nine measurement parameter usually consisting of FSC, SSC and seven colors of fluorescence. Excitation is from three spatially separated lasers (UV, 488, 560 - 640 nM tunable dye lasers) with three independent timing windows. Using the primary beam for 488 nM excitation four colors can be measured; two colors for secondary beam excitation and one color for tertiary laser excitation. This provides exceptional flexibility for combining antibody measurement with other fluorochrome combinations such as DNA dyes or for other fluorochrome measurements.
• Third laser window: We have found that coaxial beams are not practical unless emissions are of similar intensity. To eliminate this problem, we have designed a third laser window so that excitation/ emission occur at different times. This allows for resolution of fluorescence based on different excitation characteristics even if the emission spectra are identical.
• Cross beam compensation: One of the major disadvantages of two laser flow cytometry has been the assumption that this compensation would not be necessary because the fluorochromes excitation spectra would not overlap. Not so, for example, the tandem conjugates, PE-TR, PE-Cy5 or PE-Cy7, are excited by both the primary 488 nM beam (PE) and by the secondary beam (TR or Cy5, Cy7). In order to solve this problem as well as to provide for compensation flexibility for the additional parameters, we have added compensation networks to provide all possible fluorescence parameter combinations.

• High speed sorting: A major limitation of flow cytometry has been the inability to sort very large numbers of cells; usually bulk separations are performed using magnetic beads, panning techniques or specific binding columns for positive or negative selection. This may be followed by cell sorting to improve purity, but often at an unacceptable cell loss. Accordingly, we have developed a high speed cell sorter in collaboration with BDIS currently marketed as the TurboSort Option.

    There are two paradigms that need to be clearly defined when we speak of high speed sorting. The first is "high speed analysis" and slow speed sorting. This actually is the most common form referred to as high speed sorting. In reality, however, this is not high speed because the cells to be sorted are infrequent (often less that 5%) and only conventional sorting speeds are required. For example, if 1% of cells were desired, an analysis speed of 30,000 cells/sec. would result in a sorting speed of only 300 cells/sec.       This paradigm is actually high speed cell analysis to increase throughput but only conventional sorting speeds are required. This is very important for stem cells and mixed populations in which the cell of interest represents only a small fraction of the total. The second paradigm is truly high speed sorting as the events to be sorted are a significant fraction of the total (e.g. 50%). In this situation both high speed analysis and sorting are required because an analysis speed of 30,000 cells/sec. would require a sorting speed of 15,000/sec. Clearly, an instrument with this capability would be able to handle any desired frequency. The FACSVantage with TurboSort Option was conceived to meet these design features. A sort rate of 20,000 cells/sec. (7.2 x 10⁷ cells/hr) at 98% purity and 70% recovery are routine.

RESEARCH FACScan: It is now possible to perform simultaneous four color data acquisition using a single laser excitation source. The four fluorochromes used for antibody conjugation are fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-Texas Red (PE-TR) and PE-Cy5 or perdinin chlorophyll protein (PerCP). The FACScan has the capability of measuring only three of these colors. In order to accomplish four color measurements two major modifications were required. In the first, a fifth photomultiplier tube (PMT) and associated electronics were added including the necessary compensation networks. This was wholly designed and built in our electronics laboratory. In the second, the fixed filtration holders were changed to interchangeable filter holders. This provides us the flexibility of changing filters and dichroic mirrors. This instrument has been in use since 1993.

    During the design of the compensation networks, we addressed the problem of high autofluorescence associated with many cultured cell lines. This problem can be completely solved by electronic autofluorescence reduction on a cell by cell basis (Cytometry 7:566-574, 1986, Communications In Clinical Cytometry, 22:250-255, 1995). Since the Research Scan allows for changeable optical filtration, we can use one PMT for optimal detection of the desired fluorochrome and a second PMT that accepts the more broadly distributed longer wavelength autofluorescence emission that is outside the fluorochrome’s emission spectrum. The longer wavelength autofluorescence emission is subtracted from the shorter autofluorescence emission thereby nullifying autofluorescence. When the fluorochrome is present, its fluorescence is detected with optimal efficiency regardless of the cell’s autofluorescence.

Luminex FACScan: This instrument has the modifications provided by Luminex to perform multiplex immuno and molecular assays using fluorescent microspheres. In addition, the instrument can also be used for standard five parameter single laser applications.

FACSCalibur: This two laser, 6 parameter, 4 color instrument provides both clinical research and reference laboratory service. The four fluorochromes most often combined are FITC, PE, PE-CY5 (or PerCP) and APC. The entire data acquisition is automated for operator free performance.
 

 
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