1. Introduction to Flow Cytometry
IGC Workshop
General Programme
Time
Tutorial
Room
Speaker
Day 1
9h30-10h45
Fundamentals of Flow Cytometry
Ionians
Rui Gardner
11h00-13h00
Module 1 – Group 1
Module 2 – Group 2
Analyzer room
(Hands-on)
Day 2
Multicolor Analysis
Module 1 – Group 3
Module 2 – Group 1
Day 3
Applications in Flow Cytometry
Claudia Bispo
Module 1 – Group 2
Module 2 – Group 3
Day 4
9h30-11h00
Cell Sorting
11h15-13h00
FlowJo Basic Training
Giordano Bruno
Module 1 – Cell Cycle Analysis using FACScan (Cláudia Bispo)
Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)

2. Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Fundamentals of Flow Cytometry
Rui Gardner
IGC – April 2, 2013

3. Overview Definitions: What is flow cytometry and what does it measure?
Fluidics: Hydrodynamic focusing, Flow Rate...
Optics: Light, fluorescence, emission and detection...
Electronics: pulse, data acquisition...
Analysis: Tips on Data handling 3

4. Resources
Purdue Univ. Cytometry Labs: all you need to know about Cytometry!
Books:
Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version.
Tutorials:
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

5. Resources (Web Tools) BD Biosciences: 5

6. Resources (Web Tools) Life Technologies: 6

7. Resources (Web Tools) eBioscience: 7

8. Resources (Web Tools) Bioledgend: http://www.biolegend.com/webtoolstab8

9. What is Flow Cytometry used for?
Measurement of physical and chemical properties of cells.
Flow Cytometry:
Characterization of cells flowing in a stream of fluid
What is Flow Cytometry used for?
Flow cytometry can be used to count large numbers of cells or particles based on size, internal compexity, phenotype, cellular state, cell function, DNA content, gene expression, and to quantify these same cellular properties at a single-cell level.
Since the introduction of the world's first commercial as well as the first fluorescence-based flow cytometer, the ICP 11, in , key flow cytometry technologies have been pioneered by Partec. These include doublet discrimination, piezo-based closed sorting, software compensation and mobile flow cytometry systems. As a result the company holds an extensive collection of national and international key patents related to flow cytometry and cell analysis.
Flow Cytometry is not equivalent to FACS: Fluorescence Activated Cell Sorting 9

10. Key Advantages of Flow Cytometry
Population data
Measure thousands of cells/particles per second
Measure multiple parameters simultaneously.
Detection of extremely rare populations
Cell sorting
High purity, speed, and yield.
0.02% .
Flow Cytometry does not...
Locate where a certain component is within a cell
Measure distribution of cellular components
Measure cell morphology

11. Flow Cytometer in a nutshell
an illuminated volume where they
scatter light and emit fluorescence
that is filtered, collected and…
Optics
converted to digital values
that are processed
and analyzed in a computer.
Electronics
Cells in suspension
flow in single file through…
Fluidics 11

12. Flow Cytometers FACSscan (BD) MoFlo Legacy (BC) FACSCalibur (BD)
Analyzers
High Speed Cell Sorters
FACSscan (BD)
FACSCalibur (BD)
CyAn ADP (BC)
LSR Fortessa (BD)
FACSCanto (BD)
EC800 (Sony-iCyt)
Sony Spectral Analyzer (Sony)
CyFlow Cube (Partec)
MacsQuant (Miltenyi Biotec)
Guava (Merck-Millipore)
Gallios (BC)
Accuri C6 (BD)
Attune (Life Technologies)
etc
MoFlo Legacy (BC)
FACSAria (BD)
MoFlo XDP (BC)
MoFlo Astrios (BC)
Influx (BD)
FACSJazz (BD)
FACS Vantage (BD)
EPICS ALTRA (BC)
Avalon (Propel Labs)
Synergy sy3200 (sony-iCyt)
SH800 (Sony)
etc 12

13. Fluidics

14. Interrogation point 14

15. Simplified Schematics
Sheath
Flow
Sample
Injected
Laser
Flow Chamber
Sheath
Flow
Sample
Injected
Laser
Flow Chamber
Hydrodynamic
Focusing
Flow Chamber in a BD FACSAria Cell Sorter
Flow Chamber in a BD LSR Fortessa

16. Flow Rate Less cells pass per unit time More cells pass per unit time
CyAn ADP
Sheath
Flow
Sample
Laser
Low Flow Rate
Sheath
Flow
Sample
Laser
High Flow Rate
LSR Fortessa
Less cells pass
per unit time
More cells pass
per unit time
FACScan
FACSCalibur 16

17. Flow Rate Low Flow Rate High Flow Rate
Laser
Sheath
Sample
High Flow Rate
Laser
Sheath
Sample
High Power
Lower Power
High Power
Lower Power
Samples should be run at the lowest flow rate (differential pressure) as possible.
If increasing flow rate has:
No impact on population distribution: Use high flow rate.
Changes population distribution: Use low flow rate and increase cellular concentration. 17

18. Typical jet-in air cytometer
Exposure time
Typical jet-in air cytometer
Cell velocity is proportional to Sheath Pressure
𝐯= 2∆𝐏 𝜌
v ~ 5-30m/s
Laser beam height
~ 5-20 µm
𝐯 = 2∆𝐏 𝜌 𝐹
Therefore exposure time ~ s
Typically, in sorters, sheath pressure can be reduced to increase exposure time, and therefore sensitivity and resolution of acquisition.
Sheath pressure in Analyzers is usually fixed. 18

19. Optics

20. Excitation - Lasers Most common laser wavelengths available 20 488 nm
442
Most common laser wavelengths available 20

21. Multiple Laser Systems
Spatially Separated Laser Beams
Laser Intercepts
in Flow Cell
FACSAria III (BD Biosciences)
Laboratory of Molecular Immunology
Institute Molecular Genetics, Academy of Sciences, Czech Republic
FACSAria III (BD Biosciences)
Time delay between lasers must be determined for each instrument for a given sheath pressure.
Changes in Sheath Pressure will impact measurements. 21

22. Emission - Light Scatter
Light is reflected towards all directions
Low angle: Forward Scatter (FSC)
High angle: Side Scatter (SSC) 22

23. Forward Scatter (FSC)
The magnitude of Forward Scatter is roughly proportional to the size of the cell. 23

24. Side Scatter (SSC)
Side Scatter is caused by granulosity and structural complexity inside the cell. 24

25. (2D) Scatter Plot 25

26. Optics (Fluorescence)

27. Fluorescence (1) Excited State Ground State Fluorophore Energy Levels
Absorbed
Light
Emitted
Light
High Energy
Stokes
Shift
Absortion
Emission
Ground State
Low Energy
Fluorophore
Energy Levels 27

28. Fluorescence (2) Fluorophore Stokes Shift Low Energy High Energy 1 2 3
Absortion 1 3
Emission
Fluorophore
Lower Wavelength
Higher Frequency
Higher Energy
Higher Wavelength
Lower Frequency
Lower Energy

29. Excitation-Emission Spectrum (2)
Excitation maxima
Emission maxima
Each Fluorochrome has a specific Excitation and Emission Spectra

30. Excitation Spectrum (1)
Excited
480 nm
520 nm
550 nm
590 nm 30

31. Excitation-Emission Spectrum (1)
Excitation max
550 nm

32. Excitation - Emission Excitation at different wavelengths
decreases intensity of emission.

33. Fluorescence Charts

34. Fluorescence Spectra Viewers (Web Tools)
BD:
Life Technologies: Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html
eBioscience:
Bioledgend:
UArizona:
Evrogen: 34

35. Optics (Fluorescent Markers)

36. 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

37. Tandem Dyes PE-Alexa Fluor 700 PE-Cy5 PE-TxRed PerCP-Cy5.5 PE-Cy7
APC-Cy7 37

38. 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

39. Fluorescent Proteins (1)
GFP was first noted by Shimomura et al., in the jelly fish Aequoria Victoria, in 1962 and cloned 30 years later

40. Fluorescent Proteins (2)
San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins
Tsien Lab
Roger Tsien, Nobel Lectures, 2009
- PLATE - Beach.jpg

41. Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Coffee Break…!