Multicolor Flow Cytometry and Cell Sorting
Alexander Schmitz, Flow Core Manager
PhD course on Translational Cancer Research
12.-14. 11.2013 Klitgården, Skagen
This material/presentation
is
for personal use only.
Pictures have been used
without permission
And are not all
referenced properly
Pictures are taken
from the web without
Permission. Reference ?
Alexander Schmitz
How can I explain what flow cytometry is to someone that knows nothing about it?
Imagine you are visiting a supermarket.
You choose the goods you want and take them to the cashier.
Usually you have to pile them on the conveyor.
The clerk picks up one item at a time and interrogates it
with a laser to read the barcode. The items listed later on your reciept
Once identified and if sense prevails,
similar goods are collected together
e.g. Fruit and vegetables go into one shopping bag
and household goods into another.
Now picture your mind the whole process automated;
replace shopping with biological cells;
And substitute the barcode with cellular markers
- Welcome to the world of flow cytometry and cell sorting
Taken from the AbD Serotec brochure: introduction to Flow Cytometry ( by Misha Rahman et al.)
Alexander Schmitz
Basics - What is flow cytometry?
A technology that simultaneously measures
and analyzes
multiple physical characteristics
(light scatter and fluorescence)
of single particles, usually cells,
as they flow
in a fluid stream
through a beam of light (laser)
Pictures are taken
from the web.
Reference ?
Alexander Schmitz
How to get the single cells ?
Haematological Biobank
PB / BM
Intermezzo
Cell Culture & and Biobank
Homogeneous
Sample
Human normal or malignant tissues
(BM, PB; Tonsil, LN, Spleen, Thymus.)
Dilution/ Buffering
May need homogenisation
Ficoll
Cancer Cell Lines
Centrifugation
Taken from: ?
(for MM, DLBCL, FL, etc.)
Cell Counting
DMSO addition
Taken from: ?
ready to go
Taken from: ?
Freezing (Liquid Nitrogen)
Basics - Schematic overview
Cells in suspension flow in single-file (focussed) through an illuminated volume
where they scatter light and emit fluorescence
that is collected, filtered and converted to digital values that are stored on a computer.
Laser – light source to focus light
Fluidics – direct liquid stream containing
particles through focused laser beam
 Collection Optics and filters – detect light
signals coming from particles
Electronics/computer – convert light
signals to voltage and digital output
Taken from: http://flow.csc.mrc.ac.uk/?page_id=852
Alexander Schmitz
Basics - Light Scatter
Scatter Plot
Measuring scattered light...
Taken from: http://images.novusbio.com/design/lightning_5.jpg
Scattering Light
Taken from: http://www.strategic-consult.com/wp-content/uploads/2012/03/technology_pic2.jpg
Speed: Thousands of cells/second
Taken from:
http://img181.imageshack.us/img181/7652/fscxi2.gif
Alexander Schmitz
Basics - Fluorescence
Taken from: ?
Excitation
Emmision
Taken from: ?
Taken from:
http://www.chinapeptides.com/technology_t4c.php
FSC+SSC+
FL1 to FL4
= 6 parameters
from each cell
Taken from: ?
Modified from: http://prezi.com/4wqeuvsd9qff/cell-separationflow-cytometry/
Taken from:
http://131.220.28.201/wordpress/workshop-bead-based-assays-in-flow-cytometry/
Alexander Schmitz
MFC – Optics, Spectral overlap & compensation
Spectral Overlap
(Emission)
a) Optical
filters
Taken from: http://www.stemspec.ca/Project/instrument.html
Taken from: http://www.labome.com/method/
Flow-Cytometry-A-Survey-and-the-Basics.html
Taken from: http://info.ebioscience.com/bid/74887/Want-to-Simplify-the-Multicolor-Matrix-Madness
b) Compensation:
Calculations to adjust for the spectral overlaps
Taken from: https://flowcytometry.gwu.edu/sites/flowcytometry.gwu.edu/files/downloads/log.gif
Biexpontial (Logical) versus Logarythmic plot display
Taken from: http://www.flowjo.com/vX/en/gw.transform.benefits.html
History ( Flow Cytometers)
Modified from: www.cyto.purdue.edu
http://www.google.dk/url?sa=t&rct=j&q=&esrc=s&source=w
eb&cd=4&ved=0CE4QFjAD&url=http%3A%2F%2Fwww.cyto.
purdue.edu%2Farchive%2Fclass%2Fbms602a%2Flecture00
02.ppt&ei=sgGKUsiPKqjW4gTLooDYAQ&usg=AFQjCNF8q4
engdVgMpGdRMSNxY8qqQVDig&bvm=bv.56643336,d.bGE&
cad=rja
Moldovan, Andrew 1934:
.
Photo-electric technique for the counting of microscopical cells. Science 80, 188-189
Glass capillary tube mounted on microscope stage.
Gucker, Frank.T 1947:
Gucker, F.T. et al. (1947): A photoelectric counter for colloidal particles. Am. J. Chem. 69, 2422-2431
Developed a flow cytometer for detection of bacteria in aerosols
Instrument: Sheath of filtered air flowing through a dark-field flow illuminated chamber.
Light source was a Ford headlamp, PMT detector (very early use of PMT)
1969
This is newer
Wallace Coulter 1956:
The first commercial flow cytometer reached the
marketplace in 1969. It was called the ICP 11
and was distributed by Phywe, Göttingen
( Today: Partec).
measured changes in electrical conductance as cells
suspended in saline passed through a small orifice.
(Today: Beckman-Coulter)
Alexander Schmitz
State of the Art:
Flow Cytometers today
• Multiple lasers
Clinical standard: 3-4
Research: up to 6
• Increasing numbers of fluorescent
channels (detectors)
Clinical: 3-8
Research: 10 (or more) )
• Faster processing
• Digital signal processing
Compliled from various company homepages
• Automatisation
>15 detectors
• Improved software for
multidimensional analysis
and/or Easy of use
Taken from:
http://www.bioscience.co.uk/feature/135238
Taken from: http://www.bril.unsw.edu.au/facility.htm
Alexander Schmitz
Cell Sorting
Taken from: Ref: ?
Taken from:
http://ibv.unice.fr/EN/institute/facs_facility.php
Taken from:
http://www.uthsc.edu/research/research_resources/fccs/
BDFACSAriaII: Sorting with up to
8 fluorescent paramters/cell
Modified from:
http://www.mpi-muenster.mpg.de/en/research/service_groups/facs/index.html
Taken from:
http://www.gene-quantification.de/single-cell-handling.html
Alexander Schmitz
History ( Flow Sorters)
Modified from: www.cyto.purdue.edu
http://www.google.dk/url?sa=t&rct=j&q=&esrc=s&source=w
eb&cd=4&ved=0CE4QFjAD&url=http%3A%2F%2Fwww.cyto.
purdue.edu%2Farchive%2Fclass%2Fbms602a%2Flecture00
02.ppt&ei=sgGKUsiPKqjW4gTLooDYAQ&usg=AFQjCNF8q4
engdVgMpGdRMSNxY8qqQVDig&bvm=bv.56643336,d.bGE&
cad=rja
1974
Mack Jett Fulwyler, (1936-2001)
The first
Becton Dickinson
Cell Sorter (FACS)
Herzenberg, 2010
1968
Alexander Schmitz
Beyond Classical MFC
Applications
The ability to simultaneously measure multiple parameters on a cell by cell basis is probably the
most powerful aspect of analytical flow cytometry. This allows flow cytometry to be used for a wide
range of applications. Perhaps the most common use is the identification of the presence of
antigens either on the surface of or within cells. However flow cytometry may be used for the
analysis of DNA or RNA content, and for a number of functional studies on cells.
Immunophenotyping
Cell Cycle
Flowfish
Telomer Lenght (*)
Cytometric Bead Array
Apoptosis
Multicolour analysis
Fluorochromes
Quantum Dots
Principles of surface antigen analysis
Experimental design
Immunophenotyping
Phosphorylated proteins
Cytoplasmic antigen analysis
Cell counting
Cell preparation
Compensation
Viability
Cell cycle analysis
Cell cycle analysis
Cell cycle plus antigen
m phase
Bromodeoxyuridine
5-ethynyl-2-deoxyuridine
eGFP
DNA damage
PARP
Live cell cycle
Quiescence-Senescence
Replication timing
Ploidy
Imaging cell cycle
Fluorescent proteins
Flow FISH
Apoptosis
Annexin-V
Images
Caspase activity
Organelle function
DNA fragmentation
Light scatter
Autophagy
Autophagosomes
Lysosomes
Organelle Phagy
Annexin V
Membrane asymmetry
Images
Oncosis
Annexin-V
Membrane asymmetry
SYTO 16
Images
Organelle function
DNA fragmentation
Light scatter
Necrosis
Functional analysis
Calcium flux
Cell proliferation
Other assays
Rare event analysis
Dendritic cells
Stem cell/progenitor cells
Translocation assay
FRET
Bacteria
Beads - CBA technology
FlowCytoMix bead technology
Insects
Neurons
NADH
Smartflare
Muse assay kits
Taken from:
http://www.icms.qmul.ac.uk/flowcytometry/uses/
Clinical Applications of Flow Cytometry
Taken from:
Flow Cytometry in Haematology
• Historically,
Classification of lymphomas has been reliant on cytomorphology
• Today:
Multiparametric process combining
• morphological, immunophenotypic, genetetic data
• information regarding clinical presentation and disease course
Flow Cytometric Immunophenotyping important for
2008
• Determination the presence of disease at presentation
• Assisting with disease classification
• Staging of newly diagnosed malignancy
• Minimal disease monitoring
• Investigation of disease relapse or progression
Alexander Schmitz
Determination the presence of disease at presentation
Determine the presence or absence of normal
and neoplastic cell populations of differing
types by assessing:
1.
2.
3.
4.
Multiple Myeloma
Cell lineage;
Developmental stage;
Presence of clonal populations; and
A detailed immunophenotype of the population of interest
Prerequsition:
The corresponding immuno phenotype of the normal
tissue needs to be known to evaluate the presence or
absence of normal or neoplastic phenotype
Taken from: http://www.lymphomation.org/about-details.htm
Alexander Schmitz
Characterisation of corresponding normal B cell populations
in various human tissues
Modified from:
Alexander Schmitz
Alexander Schmitz
Flow cytometric immunophenotyping for hematologic neoplasms.
Taken from:
Cell Sorting in Cancer Research
Cell Sorting
Flow cytometry activated sorting
(FACS) allows:
Isolation and purification of cancer
cells for subsequent analysis
sorting of small populations (and rare
cells with frequency down to 0.03%)
Sorting single cancer cells for singlecell studies
highly selective enrichment for stem
cell autologous transplantation
(tumour free)
Circulation tumour cell analysis
Taken from: Barteneva, N.S. et al., 2013.
Cell sorting in cancer research--diminishing degree
of cell heterogeneity.
Biochimica et biophysica acta, 1836(1), pp.105–22.
Minimal Residual Disease (MRD)
(Sorting residual cells)
e.g. Multiple Myeloma
Alexander Schmitz
The future of flow cytometry in the analysis of B cell malignancies






More lasers
More fluorochromes
More simultaneously analyzed paramters
Faster Speed
Expanding capabilities ( cell size, cells“pictures”)
New antigens, especially intracellular antigens
But: Increasing complexity of flow cytometry requires:
STANDARDIZATION of diagnostic and MRD approaches:
o
o
o
o
o
Laboratory protocols (Preparation and Staining)
Instrument settings
Antibody and fluorochrome combinations
Quality standards
Advanced analysis software (multidimensional analysis )
Allows to combine data from different patients & laboratories
e.g. to create a large common database
Multicolor software analysis
Traditional analysis: ”Visual pattern recognition”
3 color experiment
(FL1, Fl2, Fl3):
1
1
2
3
2
3
Tube1: A,B,C; Tube2: A,B,D;
Tube3: A,B,E; ...
3 color experiment
(FL1, Fl2, Fl3, Fl4):
1
1
2
3
2
2
1
4
3
3
4
4
Tube1: A,B,C;D; Tube2: A,B,E,F; Tube3: A,B,G,H; ...
Orfao et al., ESCCA course presentaion 2010
Alexander Schmitz
8 color experiment
(Fl1-Fl8):
Alexander Schmitz
Multicolor software analysis
 A) Need for more visual datapresentation
B) Need for fast, objective and multidimensional dataanalysis
Infinicyt
Beckman-Coulter, Kaluza
Infinicyt MFC analysis software
Basic Gating Feature Demo
Taken from: infinicyt.com
Pictures are taken
from the web without
Permission. Reference ?
Alexander Schmitz