Introduction to Flow Cytometry
IGC Workshop
April 27-30, 2010
General Programme
Day 1
Day 2
Day 3
Day 4
Time
Tutorial
Room
Speaker
9h30-10h45
Fundamentals of Flow Cytometry
Ionians
Rui Gardner (IGC)
11h00-13h00
Policies and procedures when using the IGC
FACS equipment
FACS room
Telma Lopes (IGC)
9h30-10h45
Multicolor Analysis
Ionians
Rui Gardner (IGC)
11h00-13h00
Module 1 – Group 1
Module 2 – Group 2
FACS room
9h30-10h45
Applications in Flow Cytometry
Ionians
11h00-13h00
Module 1 – Group 2
Module 2 – Group 3
FACS room
9h30-10h45
Cell Sorting
Ionians
11h00-13h00
Module 1 – Group 3
Module 2 – Group 1
FACS room
Module 1 – Cell Cycle Analysis using FACScan (Telma Lopes)
Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)
Rui Gardner (IMM)
Telma Lopes (IGC)
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Fundamentals of Flow Cytometry
IGC – April 27, 2010
Overview
• Definitions: What is flow cytometry and what does it measure?
• Fluidics: Hydrodynamic focusing
• Optics: Light, fluorescence, emission and detection
• Electronics: pulse, data acquisition
• Analysis: Data handling
3
Resources
Purdue Univ. Cytometry Labs: all you need to know about Cytometry!
• http://www.cyto.purdue.edu
Tutorials:
• http://probes.invitrogen.com/resources/education
Spectra Viewers:
• http://www.bdbiosciences.com/spectra
• http://probes.invitrogen.com/resources/spectraviewer
• http://www.mcb.arizona.edu/ipc/fret/
Books:
• Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version.
http://probes.invitrogen.com/lit/practicalflowcytometry/toc.html
http://www.coulterflow.com/
History of Flow Cytometry:
• Shapiro, H. (2007) “Cytometry and Cytometers: Development and Growth”,
in Flow Cytometry with Plant Cells (Eds, J. Dolezel, J. Greilhuber, J. Suda),
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4
What is Flow Cytometry?
Cytometry:
Measurement of physical and chemical properties of cells.
Flow Cytometry:
Characterization of cells flowing in a stream of fluid
Fluidics
Cells in suspension
flow in single file through
Optics
an illuminated volume where they
scatter light and emit fluorescence
that is filtered, collected and
Electronics
converted to digital values
that are processed and analyzed in a computer
FACS: Fluorescence Activated Cell Sorting
5
Key Advantages of Flow Cytometry
• Population data
• Measure thousands of cells/particles per second
• Measure multiple parameters simultaneously.
• Detection and sorting of extremely rare populations
0.02%
• Cell sorting
• High purity, speed, and yield.
Flow Cytometers
FACSCalibur
FACSscan
FACSCanto
LSR II
CyAn ADP
etc
FACSAria
MoFlo
FACS Vantage
EPICS ALTRA
etc
7
The Instrument
8
Interrogation point
10
Hydrodynamic Focusing
Low Flow Rate
Sheath
Flow
Sample
Flow
Sheath
Flow
Laser
High Flow Rate
Sheath
Flow
Sample
Flow
Sheath
Flow
Laser
Less cells pass
per unit time
More cells pass
per unit time
11
Flow Rate
High Flow Rate
Low Flow Rate
Laser
Sheath
Laser
Sheath
Sample
Sample
3105
3105
2328
1552
Counts
Counts
2328
1552
776
776
0
100
101
102
FL2 Log Comp
103
104
0
100
101
102
FL2 Log Comp
103
104
12
Lasers
488 nm
Most common laser wavelengths available
14
Light Scatter
Light is reflected towards all directions
Low angle: Forward Scatter (FSC)
High angle: Side Scatter (SSC)
16
Forward Scatter (FSC)
The magnitude of Forward Scatter is roughly
proportional to the size of the cell.
17
Side Scatter (SSC)
Side Scatter is caused by granulosity and
structural complexity inside the cell.
18
(2D) Scatter Plot
19
Fluorescence (1)
Excited State
Absorbed
Light
High Energy
Emitted
Light
Ground State
Low Energy
21
Fluorescence (2)
High Energy
2
2
1
1
3
3
Low Energy
Excitation Spectrum (1)
480 nm
520 nm 550 nm
590 nm
Excited
23
Excitation-Emission Spectrum (1)
Excitation
Excitation max
550 nm
Emission
Excitation - Emission
Excitation at different wavelengths
decreases intensity of emission.
Excitation-Emission Spectrum (2)
Excitation maxima
Emission maxima
Fluorescence Charts
Fluorescence Spectra Viewers
Invitrogen (http:// www.invitrogen.com/spectraviewer)
BD Biosciences(http:// www.bdbiosciences.com/spectra)
Reactive and Conjugated Probes
Cascade Blue
Pacific Blue
Pacific Orange
Alexa Dyes
Lucifer yellow
NBD
R-Phycoerythrin (PE)
PE-Cy5 conjugates
PE-Cy7 conjugates
PE-Texas Red
PerCP
TruRed
PerCP-Cy5.5 conjugate
Fluorescein (FITC)
BODIPY-FL
TRITC
X-Rhodamine
XRITC
Lissamine Rhodamine B
Texas Red
Allophycocyanin (APC)
APC-Cy7 conjugates
Etc…
Immunohistochemistry
Direct method
Indirect method
First dye-coupled antibody using fluorescein isothiocyanate
(FITC) patented by Joseph Burckhalter and Robert Seiwald,
in 1960
Pictures adapted from wikipedia
Tandem Dyes
PE-Alexa Fluor 700
PE-Cy5
PE-TxRed
PerCP-Cy5.5
PE-Cy7
APC-Cy7
31
DNA and Cell Function Probes
Calcium
Membrane
pH
Cell Tracker
DNA
Mitocondria
Fura 2
FM 1-43
SNAFLE
CMRA
DAPI
Nonyl Acridine O
Indo 1
FM 4-64
SNARF
CMTPX
HOECHST
MitoSox
Bapta/dye
DiO
BCECF
BMQC
TOPRO3
Mitotracker
Calcium Green
ANNINE 6
pHRodo
DMFDA
PI
Rhodamine 123
Fluo 4
Di4ANNEPS hq
lysosensor
CMTMR
DRAQ5
MitoProbe
Fluorescent Proteins (1)
GFP was first noted by Shimomura et al., in the jelly fish Aequoria
Victoria, in 1962 and cloned 30 years later
Fluorescent Proteins (2)
San Diego beach scene drawn with living
bacteria expressing 8 different colors of
fluorescent proteins
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Coffee Break…!