Current Research Applications of Flow Cytometry and Cell J.Paul Robinson Professor of Immunopharmacology Professor of Biomedical Engineering Purdue University Email: [email protected]WEB: http://www.cyto.purdue.edu ty Lecture at Kitasato University, Towada, Japan 26-July 4, 2000
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Current Research Applications of Flow Cytometry and Cell J.Paul Robinson Professor of Immunopharmacology Professor of Biomedical Engineering Purdue University.
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Current Research Applications of Flow Cytometry and Cell
J.Paul RobinsonProfessor of Immunopharmacology Professor of Biomedical EngineeringPurdue UniversityEmail: [email protected]: http://www.cyto.purdue.edu
Faculty Lecture at Kitasato University, Towada, JapanJune 26-July 4, 2000
Lecture summaryThis lecture will discuss the principles of flow cytometry and how they are applied to basic research and clinical questions. We will discuss the general principles of how a flow cytometry operates and why this technology has advantages over many others. In addition, we will look as some examples of newer applications such as apoptosis, multiplexed bead assays and future applications. Cell sorting using your recently acquired Coulter Altra will be described and the key features discussed.
Purdue University Cancer Center&
Purdue University Cytometry Laboratories
What can Flow Cytometry Do?
• Enumerate particles in suspension• Determine “biologicals” from “non-biologicals”• Separate “live” from “dead” particles• Evaluate 105 to 106 particles in less than 1 min• Measure particle-scatter as well as innate fluorescent• Measure 2o fluorescence• Sort single particles for subsequent analysis
Introductory Terms and Concepts Parameter/Variable
Coulter Optical System – Elite/AltraCoulter Optical System – Elite/Altra
• The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted
555 - 595
PMT4
APC 655 - 695
PMT6
PMT7
49
0
DL
488
BK
05
5
DL
62
5
DL
675
BP
488 BP525 BP575 BP
Purdue Cytometry Labs PUCL3034
632
BP
TM
PMT3 PMT2 PMT1
PMT5
Fluidics
SMALL BEAD LARGE BEAD
Frequency Histogram
SMALL BEAD LARGE BEAD
Sample inSheath
Sheath in
Laser beam
Stream Charge
+4KV -4KV
Waste
SORT RIGHTSORT LEFT
SORT DECISIONS
Piezoelectriccrystal oscillator
Last attacheddroplet
LEFT RIGHT
Sensors
Sensor
Signals are collected from several sensors placed forward or at 90° to the laser beam. It is possible to “sort” individual particles. The flow cell is resonated at a frequency of approximately 32KHZ by the piezoelectric crystal mounted on the flow cell. This causes the flowing stream to break up into individual droplets. Gating characteristics can be determined from histograms (shown right) and these can be used to define the sort criteria. These decisions are all controlled by the computer system and can be made at rates of several thousand per second.
Figure 1 The central component of a flow cytometer is the flow cell. A cutdown of a typical flow cell indicates the salient features. Sample is introduced via the sample insertion rod. Sheath fluid (usually water or saline) is ntroduced to surround the insertion rod causing hydrodynamic focussing of flowing cells which are contained within a core fluid. The laser intersects the fluid either outside the flowcell (in air) or in a slightly extruded portion of the flow cell tip (in quartz).
Fluorescence• The wavelength of absorption is related to
the size of the chromophores
• Smaller chromophores, higher energy (shorter wavelength)
Fluorescence• Stokes Shift
– is the energy difference between the lowest energy peak of absorbance and the highest energy of emission