Instrument Description
The FACS Vantage allows the isolation of defined cell subset(s) from heterogenous mixtures. Cells can be sorted according to size, granularity, surface markers (up to 3 color fluorescence (5 with SE upgrade)) and DNA content at a speed of 8,000 – 10,000 cells per second. Chromosomes and non-cellular particle sorting are also possible. An advantage of using the FACS Vantage is that cells can be sorted sorted at rates up to 25,000 cells/second in a sterile environment enabling the recovered cells to be cultured. This is especially applicable to transfected cells, where only a small proportion of the cells may express the antigen of interest.
Equipment Specification and Features
ü Manufactured August 2001
ü Ugradeable to FACS Vantage SE
ü Coherent Enterprise II Laser Modell 621
ü Enterprise Laser Support Kit
ü Coherent Enterprise LP5I Wärmetauscher
ü Pressure/Vacuum pump
ü FL-1 PMT for Laser 2
ü FL-2 PMT for Laser 2
ü INDO-1 Filter Set
ü Clone CYT with Indexsorting
ü Cooling system
ü FACS-Vantage™ incl.
ü SEM, TurboSort, MacroSort, Accudrop
ü Computer FACStation Silver G4 (Macintosh)
ü BD PAC-Kit
ü 15’’ LCD Monitor
ü 18’’ LCD Monitor
ü Graphic card for 2-monitor display
ü Printer Tektronix Color 850 N
ü Cell Quest Software Pro V3.3 with USB-Key
Methods
The FACS Vantage uses electrostatic droplet sorting by the generation of droplets from the core stream. This produces a stable line of droplets at 10,000 to 60,000 Hz, depending upon nozzle size. Larger cells use a larger nozzle and lower drop-drive frequencies and so are sorted at lower optimum rates. To prevent distortion of the laminar flow of the sample/sheath fluid and disruption of the stable droplet break-off (clogging of the Nozzle), it is required that ALL samples be filtered prior to sorting. Sterilized nylon mesh (20 - 70 uM pore size) can be used. The number of cells that can be sorted depends upon factors such as frequency of events to be sorted, total number, and required purity. These factors can be discussed prior to sorting. Sheath fluid used is sterile 0.9% Sodium Chloride or PBS. Sterilization of the cell sorter is performed as required using 10 % bleach, 70 % ethanol and distilled water.
The FACS Vantage allows the isolation of defined cell subset(s) from heterogenous mixtures. Cells can be sorted according to size, granularity, surface markers (up to 3 colour fluorescence) and DNA content typically at a speed of 8,000 – 10,000 cells per second. Chromosomes and non-cellular particle sorting are also possible.
What makes Flow cytometry such a powerful technique, is its ability to measure several parameters on many thousands of individual cells in a very short period of time, by the measurement of their fluorescence and the way they scatter light. This involves shining a laser on discrete groupings of cells. The light passes through the group (forward scatter) or reflects off it (side scatter). The side scatter and forward scatter are measured by photodetectors. Most of the time, fluorescent material(s), tuned to the wavelength of the laser, is/are selectively bound to the cells. The emitted light is filtered out through the use of wavelength-specific light filters, which transmits certain wavelengths of light and reflects others. The reflected light is turned to an electrical signal by photomultiplier tubes (PMTs). All of this data is sorted out by a computer, and plotted onto the computer screen. With a little practice and knowledge, one can tell different types of cells by the characteristics of the data produced.
Primary laser fluorochromes (excitation at 488 nm)
Fluorochrome Emission
FITC 525nm
PE 575nm
PE-TexasRed (Red613) 613nm
GFP 515nm
Propidium Iodide 600nm
Second laser fluorochromes (excitation at 351-364 nm)
Fluorochrome Emission
Cascade Blue 420 nm
Hoechst 465 nm
DAPI 465 nm
AMCA 460 nm
Third laser fluorochromes (excitation at 568-647 nm)
Fluorochrome Emission
Texas Red 613 nm
CY5 675 nm
APC 660 nm