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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
• Light Scatter- Forward (FALS), narrow (FS) - Side, Wide, 90 deg, orthogonal
• Fluorescence - Spectral range
• Absorption - loss of light
• Time - Kinetics
• Count - number of events/particles/cells
Concepts
Scatter: Size, shape, granularity, polarized scatter (birefringence)
Fluorescence: Intrinsic: Endogenous pyridines and
flavinsExtrinsic: All other fluorescence
profiles
Absorption: Loss of light (blocked)Time: Useful for kinetics, QCCount: # events -always part of any collection
Instrument Components
Electronics: Control, pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control
Optics: Light source(s), detectors, spectral separation
Fluidics: Specimen, sorting, rate of data collection
Data Analysis: Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation
Arc Lamp Excitation SpectraIr
rad
ian
ce a
t 0.
5 m
(m
W m
-2 n
m-1)
Xe Lamp
Hg Lamp
Shapiro p 99Shapiro p 99
Lasers• Argon laser
• He-Ne Laser
Optical Collection systems
He-Cd Laser Argon Laser He-Ne Laser2nd Argon Laser
Elite Cytometer with 4 Lasers
Water cooled argon laser
He-Cd laser
Air-cooled argon laser
Santa clause
Optical DesignOptical Design
PMT 1
PMT 2
PMT 5
PMT 4
DichroicFilters
BandpassFilters
Laser
Flow cell
PMT 3
Scatter
Sensor
Sample
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
495 nm 520 nm
Stokes Shift is 25 nmFluoresceinmolecule
Flu
ores
cnec
e In
tens
ity
Wavelength
Ethidium
PE
cis-Parinaric acid
Texas Red
PE-TR Conj.
PI
FITC
600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines
Fluorescence
Resonance Energy Transfer
Inte
nsi
ty
Wavelength
Absorbance
DONOR
Absorbance
Fluorescence Fluorescence
ACCEPTOR
Molecule 1 Molecule 2
Flow cytometry measurements
L
M
G
SCATTER FLUORESCENCE IMAGE
Data Presentation Formats
• Histogram• Dot plot• Contour plot• 3D plots• Dot plot with projection• Overviews (multiple histograms)
Data Analysis Concepts
Gating • Single parameter• Dual parameter• Multiple parameter• Back Gating
Note: these terms are introduced here, but will be discussed in more detail in later lectures
FITC Fluorescence
Mo1
CD4 CD8
CD8
CD45
leu11a
CD20 Tube
ID
The Cell Cycle
G1
MG2
S G0Quiescent cells
A DNA histogram
G0-G1
S
G2-M
Fluorescence Intensity
Cel
l Num
ber
A typical DNA Histogram
G0-G1
S
G2-M
Fluorescence Intensity
# of
Eve
nts
log Thiazole Orange.1 1000 100 10 1
Count
0
150
112
75
37
RMI = 0RMI = 0
log Thiazole Orange.1 1000 100 10 1
Count
0
150
112
75
37
RMI = 34RMI = 34
Reticulocyte Analysis
Labeling Strand Breaks with dUTP [Fluorescein-deoxyuridine triphosphate (dUTP)]
Green Fluorescence is Tdt and biotin-dUTP followed by fluorescein-streptavidinRed fluorescence is DNA counter-stained with 20µg/ml PI
PI-Red Fluorescence
Green Fluorescence
Green Fluorescence
Sid
e S
catt
er
Forward Scatter
Green:apoptotic cells
Red:normal cells
R2: Apoptotic Cells
R1: Normal Cells
Scatter Pattern of Human leukocytes
Lymphocytes
Monocytes
NeutrophilsA flow cytometryscattergram
For
war
d sc
att e
r (s
ize)
Side scatter (granularity)
Three Color Lymphocyte Patterns
CD3
CD
4
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD4 -->
CD3
CD
4
CD8C
D8
10 1 10 2 10 3 10 4
CD8 -->
101
102
103
104
CD4 -->
10 1 10 2 10 3 10 4
CD3 -->
101
102
103
104
CD8 -->
Data from Dr. Carleton Stewart
YoYo-1 stained mixture of 70% ethanol fixed E.coli cells and B.subtilis (BG) spores.
mixture
BG E.coli
BG
E.coli
mixture Run on Coulter
XL cytometerS
catt
er
Fluorescence
Sca
tter
Live cell/dead cell PI Fluorescence
Data from Dr. Doug Redelman, Sierra Cytometry
PIHoechst 33342
Oxidative Reactions
• Superoxide Hydroethidine• Hydrogen Peroxide
Dichlorofluorescein• Glutathione levels Monobromobimane• Nitric Oxide Dichlorofluorescein
Calcium Flux
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 50 100 150 200
Rat
io: in
tens
ity o
f 46
0nm
/ 4
05nm
sig
nals
Time (seconds)
Time (Seconds)0 36 72 108 144 180
RATIO [short/long]
0
200
400
600
800
StimulationStimulation
Flow Cytometry Image Cytometry
Membrane Potential• Oxonol Probes • Cyanine Probes
How the assay works:• Carbocyanine dyes released into the surrounding media as cells depolarize
• Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs
Time (sec)
Gre
en F
luor
esce
nce
Repolarized Cells
051
210
24
0 300 1500 1200 2400Time (sec)
051
210
24G
reen
Flu
ores
cenc
e
PMA Added fMLP Added
Depolarized Cells
Summary
Main Applications
• DNA and RNA analysis
• Phenotyping
• Cell Function
• Sorting and cell isolation
• Immunological assays
The facilities at Kitasato University
Coulter AltraThe facilities at Kitasato University
Coulter XL CytometerThe facilities at Kitasato University
