General Technique
Cytometers and cell sorters use laser excitation to characterize the scattering and fluorescence from cells and other particles. Cells in solution are hydrodynamically focused to pass one by one through a focused laser. From the amplitude of the scattered laser light, one may infer the relative size and granularity of the cell in the laser beam. In addition, cytometers can analyze cells based on their fluorescent signature. Cells may contain endogenous fluorophores, such as chlorophyll or phycoerythrin, or they may be characterized by their ability to localize fluorescent antibodies or fluorescently labeled DNA. The inFlux cell-sorting platform was developed at the ISB and has the capability to use up to three lasers in succession to simultaneously excite and characterize many fluorophores. Cell sorters differ from cytometers in their ability to separate cells of interest from a complex mixture. Once a cell has been cytometrically characterized, the sorter uses a combination of electronic delays, electrostatic charging, and a static electromagnetic field to separate the chosen cell from the other cells in solution.
The ISB has two high-speed cell sorters, a Miltenyi autoMACS magnetic cell sorter, and a FACSCalibur cytometer, in its cytometry facility.
Purpose/use/application of the technique:
Cell sorting is used to isolate cells from complex mixtures. The cells that are isolated may be discriminated based on a number of characteristics including size, shape, DNA content, or affinity for fluorescent ligands. Because the cell sorter has the ability to characterize more than a million cells a minute, one can screen large populations for the presence of rare cells. Cell sorters also may be used as a sophisticated deposition system. The electrostatic plates that control deposition of the sorted fraction can handle a number of formats including mini centrifuge tubes, as well as 96-well and 384-well plates.
Example(s) of projects at ISB that use this technique:
- Sorting of clinical prostate epithelial cells based on surface cell markers for the isolation of prostate stem cells (Hood Lab/Liu)
- PCR analysis of mRNA isolated from single cells to characterize cell to cell variation in an otherwise homogenous population (Ozinsky Lab)
- Sorting of transformed single cells based on GFP expression to create monoclonal populations (Aderem Lab/Diercks)
- Sorting reactive-oxygen-producing macrophages for analysis using rPCR and microarrays (Underhill)
- Panning a fluorescently labeled phage library against marine phytoplankton for the isolation of ScFv antibodies (van den Engh Lab/Orellana)
- Purification of human pancreatic ß Cells based on auto-fluorescence (Kutlu /JDRF)
Ongoing area of technology development:
We continue to make improvements to the high-speed cell sorters to either extend their capabilities or improve the purity or recovery rate of the sorts. A few projects that highlight some of these innovations are:
1) a method for reconstructing the complete emission spectra of cells analyzed in the flow cytometer. 2) a means for measuring nanometer-size perturbations on the surface of a liquid capillary. This latter invention helps monitor the transition of the column of fluid into droplets, a process whose stability is critical for high-speed cell sorting.
Representative publication(s):
Gold, E.S., R.M. Simmons, T.W. Petersen, L.A. Campbell, C.C. Kuo, and A. Aderem 2004. Amphiphysin Iim is required for survival of Chlamydia pneumoniae in macrophages. J.Exp.Med. 200: 581-586.
Lansdorp, B.M., P.I. Nelson, T.W. Petersen, and G. van den Engh 2004. An optical monitor for measuring the amplitude and phase of oscillations on the surface of a capillary jet in a high speed cell sorter. Review of Scientific Instruments 75: 741-748.
Petersen, T.W., S.F. Ibrahim, A.H. Diercks, and E.G. van den 2004. Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342. Cytometry 60A: 173-181.
Petersen, T.W. and G. van den Engh 2003. Stability of the breakoff point in a high-speed cell sorter. Cytometry 56A: 63-70.
Uy, J.L., C.L. Asbury, T.W. Petersen, and G. van den Engh 2004. The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules. Cytometry 61A: 18-25.
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