Recent Advances in Multi-color Flow
Cytometry Applications
John Daley
Director of Flow Cytometry
Hematalogic Neoplasia
Dana-Farber Cancer Institute
Harvard Medical School
john_daley@dfci.harvard.edu
Recent Advances and Applications at
HemNeo Flow
http://research.dfci.harvard.edu/flowlab
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Addition of FACSAria Special Order Research Platform (SORP)
Cytometer July 2006
Ultraviolet Laser upgrade January 2007: New Options
Calcium Flux on T cell subpopulations: Tips and Tricks
Assay integration
– Stem cells : Cell Cycle/ ROS/ Apoptosis
– GFP+DsRed Co-expression with viable cell cycle: HO33342
Side Population analysis and sorting: Worth a second look
Multicolor Treg analysis and Sorting : A new way to gate
Multicolor Stem Murine stem cell sorting: Checking compensation
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Rare event Sorting: Instrument considerations , Haploid sperm cells,
Human Tetramer Sorting, Single cell PCR cloning
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Addition of FACSCanto II : switching to plates and analyzing by batch
FACSAria Special Order
Research Platform (SORP)
UV Laser: 20mW 355nm
UV PMT TRIGON
SORP OPEN OPTICAL BENCH
UV Applications
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Viable Cell Cycle sorting using Hoechst
33342
Calcium Flux using INDO-1 AM
Side Population Analysis (SP): HO33342
Live/Dead exclusion using DAPI
Apoptosis using Hoechst 33342 and PI
(future)
Quantum Dot Excitation (future)
FACSAria SORP UV Laser Upgrade January 2007
GFP+DsRed/Hoechst 33342
Viable Cell Cycle
Assay Integration
Hoechst Blue=450BP filter
GFP=FITC 530 filter
DsRed=PE 575 filter
Ca+ flux with Indo 1-AM
Useful to study heterogeneity within
Defined subpopulations
-Ratio metric Dye: Normalizes for cell size variability
As well as laser power fluctuations over time
-Very sensitive to slight shifts within populations
-Ultraviolet excited can be used with many visibly excited
-fluorochromes
FACSAria: UV Applications
Calcium Flux Kinetic Assays
Ionomycin
Calcium Flux
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Method to get Gap in Time vs. ratio
Histogram ( Lift Cover/reset time
stop/append file/close cover after 5
seconds)
Indo-Violet=405nm BP Filter
Indo-Blue=530nm BP Filter
Dichroic=505nm LP
FACSAria “SORP”
20Mw
355nm UV Laser
10ug
GAM
IgG1
0 minutes 5
Ionomycin
Calcium Flux on T cell subpopulations
Side Population (SP)
Analysis and Sorting
Background History
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Described by Margaret (Peggy) Goodell while in Richard
Mulligan's Lab at MIT in Boston
Most of flow cytometry work done by Glenn Paradis: currently
Director of Flow at M.I.T.
Looking for quiescent cells in Bone Marrow
Instrument Limitations 3 colors used in blue
UV used for Hoechst cell cycle Analysis and PI viability
exclusion ( PMT constraints)
Done on BD FACSVantage
Run at low pressure, Cell cycle resolution of lymphoid
subpopulations possible
Observed distinct side hook population profile
Phenotype determination and sorting followed by repopulation
experiments established SP identity
Howard Shapiro
Surprise 65th
Birthday party
Glenn Paradis
(did flow at MIT on
original
SP paper)
There is life other than
flow!
Empire Garden Chinatown
Boston Nov 2006
Photos by Akos Silivasi
Side Populations (SP)
by Flow
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Stringent Protocol Adherence
Instrument Parameters Critical
Tight C.V.’s (co-efficient of Variation)
Optimize Optical Filter selection
Instrument Alignment Conditions
UV Bead Standard
Fluoresbrite® BB Carboxylate Microspheres 4.5µm
Polysciences
LOG
LINEAR
Hoechst Blue
PMT= 225 Volts
Stem cell Phenotype Integration with Multiple Assays
FoxOs Are Critical Mediators of Hematopoietic Stem Cell
Resistance to Physiologic Oxidative Stress
Cell, Vol 128, 325-339, 26 January 2007
Side Populations
Instrument Parameters
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Hoechst 33342(HO) excited by Ultraviolet (U.V.) or
Violet lasers
DyeCycletm Probes excited with violet, blue or red
lasers
Optical filters 450nm for Blue H0em
670 LP for Red HOem
(PI for viability ex with uv or blue Laser)
(Optional cell surface markers for phenotypic analysis)
SP Labeling procedures
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Verify technique by using same strain and EXACT rigorous conditions
Found in above mentioned protocol
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Maintain incubation precisely at 37 centigrade
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Use C57BL/6 mice bone marrow 5-8 weeks of age for initial analysis
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Count cells accurately and resuspend at 106/ml in pre-warmed DMEM
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Add Hoechst at a final conc. of 5ug/ml (200x dilution of stock)
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Mix cells place in water bath exactly for 90 min. mix tube
during incubation
SP Labeling procedures
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After 90 minutes incubation spin cells down in the COLD and resuspend in
Cold HBSS+
Run on Cytometer or stain with antibodies or magnetic bead
depletion procedures . All steps MUST be performed at 40C
Use C57BL/6 mice bone marrow 5-8 weeks of age for initial analysis
Count cells accurately and resuspend at 106/ml in pre-warmed DMEM
Add Hoechst at a final conc. of 5ug/ml (200x dilution of stock)
Mix cells place in water bath exactly for 90 min. mix tube
during incubation
Side Population enumeration using Hoechst 33342
SP Phenotype
Murine Bone Marrow
Ho33342 dye uptake kinetics
- incubation time and concentration titration important
Violet Excitation SP
HO33342 BM
+ Verapamil
0.4%
0.3%
PMT blue= 550
PMT Red= 944
William Telford
N.I.H.
Spectral
Viewer
Ultraviolet 20mW 355nm
SP HO33342 BM
HO BLUE (450)
+ Verapamil
4.5%
0.2%
HO RED (670)
PMT blue= 316
PMT Red= 584
Other Interesting
Developments
Multiple Gating strategies
help identify low frequency
functional subpopulations
Multicolor(5) Minor populations : Four way sorting:
Treg Story
CD45ra FITC
CD127 PE
CD25 PE-CY5
CD4 PE-CY7
CD3 PAC BLUE
Four way reg sort
Treg Post Sort Reanalysis
POST SORT REANALYSIS: HUMAN PBL CD3+CD4+ CD45RA+ &CD45RATreg+/Teffector+/ --
99.4
98.4
97.1
99.9
FoxP3 Expression in CD4+
Treg CD45RA+
Fluorescence minus 1
Rare Events
The Needle in the Haystack Story
There are many ways to approach the needle in the haystack problem:
•A known needle in a known haystack
•A known needle in an unknown haystack
•An unknown needle in an unknown haystack
•Any needle in a haystack
•The sharpest needle in a haystack
•Most of the sharpest needles in a haystack
•All the needles in a haystack
•Affirmation of no needles in a haystack
•Things like needles in any haystack
•Let me know whenever a new needle shows up
•Where are the haystacks?
Burn the Haystack
Rare Events and Flow Cytometry
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Minimal residual disease (MRD)
GFP top 0.1%
Outliers
PBL stem cells
Bone marrow stem cells
Polychromatic subpopulations
Tetramers: Ag Specific T cells
1N pre Sperm cells : G. Daley
GFP 0.7%- 99% 20,000
sec
PRESORT
POST SORT
Rare Event Tetramer Sorting
0.007%
bead auto compensation
Title: Sorting Tetramer positive T cells on FACS Aria
Staining:
1.
2.
3.
4.
5.
Obtain 42 mL of whole blood from donor 430-TW56-5643
Ficoll PB, wash, and count 35.0 Million PBMC.
Resuspend in 200uL PBS/FBS
Add Tetramers as shown below:
Incubate for 30min at RT.
6. Add Abs as follows:
Tube
No.
FITC
uL
PE
u
L
ECD
u
L
PC5
u
L
PC7
u
L
APC
u
L
PacBlu
u
L
Sort
CD4/14/
19/56
8/16/
4/8
EBV
Tet
5
CD45RA
8
CD62L
8
CD8
8
Flu
Tet
5
AnnV
5
7. Incubate at RT for 30min.
8. Wash, resuspend in 500uL of AnnexinV binding buffer and place on ice.
9. Make sure to let cells sit on ice at least 1 hour prior to sorting for proper tetramer binding.
10. 15 minutes prior to sorting (or during setting of compensation) add 5uL of AnnexinV to
cells sitting on ice.
11. After incubation add 1.5 mL of AnnexinV buffer.
12. Store cells on ice until ready to sort.
Sorting
13. Add ~500uLof RNase-free PBS to each siliconized-eppendorf collection tube.
14. Strain cells twice and sort cells.
Tetramers: Ag Specific T cells
Small events : bright label
heavy gating
Rare Events and Flow Cytometry
Practical Considerations
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Number of events acquired increase
for statistical accuracy
Controls reduce number of cells
available for analysis/sorting
Reanalysis of sorted populations
conundrum
Proper Controls
Is it real or an artifact?
Rare Events and Flow Cytometry
Instrument considerations
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Optimal alignment
Tight side streams
Deflection channel choice
Take some neg to get stream
Add carrier bead
2x sort for enrichment /purity
Collection vessel options
Gating strategy
Rare Events and Flow Cytometry
Experimental Design Strategy
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Create artificial sample that mimics
real life scenario (Spike , beads and
Cells) MRD, Stemtrol,
Employ Image to visualize desired
subpopulations post sort
Recertify artificial situation post sort
Judiciously clean instrument and
measure background particle value
4 way Bead sort to check
Instrument Accuracy
Protocol
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Beads needed
1: Accudrop: cat#345249
2: Calbrite APC: cat #340487
3: Sphero Rainbow Fluorescent particles 3.0-3.4um(mid range FL1) cat# 556298
4: "
"
"
"
"
( brighter?)
cat# 556291
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First : click on new experiment icon and used default pmt setting 250, 300, 500,etc.....
and made single graphs of all pmts log except FALS and two parameter of Hoechst blue vs. APC Cy7
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Second: Add a few drops of each bead to separate tube add 1xpbs(200ul)
10,000events
and run/ record 5,000-
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Third:
Adjust APC Cy7 down to 385 volts when run Accudrop to get beads on scale. Use
Biexponential display option
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Four: mix beads together one at a time and run and gate on where peak showed up on apccy7 vs.
hoechst blue histogram (that way can see where each bead size scattered based on color gating)
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Fifth :mixed all beads together and create four distinct sort regions on APC-CY7 vs. Hoechst Blue
graph and did a four way sort for about 4 minutes sorted about 1.2 million. Use custom sort precision
settings which is : 0 32 0 (VERY VERY IMPORTANT!) I did not have them very concentrated and sorted
at 11 flow rate (bad for core stream)
reanalyze each fraction with a pbs wash between each tube.
Simple Four Way bead Sort
4 way bead sort Spiked with Fixed Murine Spleen cells
Presort Frequency
Isolation of minor subpopulations
G. Daley Lab (Melissa H)
Mouse embryonic stem cells/ Sperm cell Precursors
Nature: December 10, 2003
Picture credit: Niels Geijsen
Rare Event Gating Strategy : Pre Sperm
RT-PCR Single Cell Cloning
One is one
The ultimate rare event
Thermocycler Plate
Bottom cut off to fit in
ACDU unit
Sophia Adamia
Ken Anderson Lab
DFCI
Adjust stream
not plate
Flash drive cover
Used as wedge
Check to see if Green beads on bottom
Of vial not sidewall
10uL lysis Buffer
RT-PCR by Flow Cytometry
Waldenstrom’s macroglobulinemia
CD19+ (B cells ) from patient bone marrow aspirate
1. PCR Reaction: Thermocycler
2. Capillary electrophoresis on ABI DNA Genetic Analyzer
DNA Fragment Analysis
Capillary electrophoresis
ds DNA
3’
5’
5’
3’
Fragment flow
through capillary
DENATURE
ss DNA
3’
3’
5’
5’
5’-primer
DETECTION
WINDOW
3’-primer
5’
3’
5’
3’
5’
ELECTROKINETIC
INJECTION
3’
SAMPLE
DENATURE
Expression of SIVA splice variants in healthy donor BM CD19+B cells
Murine Hematopoietic Stem Cell/Progenitor
Sort
Koichi Akashi Lab
DFCI
Sort purpose : Gene Analysis for PCR amplification
Method: 1.Extract cells from Bone marrow (5 mice)
2.Enrich target cells by depleting Lineage Positive cells using rat
antibodies specific for lineage markers such as T-cell, B-cell,
granulocyte/macrophage and erythrocyte.
For sorting HSCs & CLP, Lin markers include CD3, CD4, CD8,
CD19, B220, Gr-1, Ter119.
3.Magnetic bead deplete Lin+ cells using sheep anti-rat coated
magnetic beads and collect negative fraction
4.Label with appropriate antibodies for HSC, CLP,
or Myeloid progenitors
Murine Hematopoietic Stem Cell/Progenitor Sort
Staining strategy
HSCs and CLPs: FITC-Sca-1
bioinylated IL-7R+PE SAV
APC-c-Kit
Myeloid progenitors: FITC CD34
PE-Fcg RII/III
APC-c-Kit
PE-Cy7-Sca-1
Add Propidium Iodide (PI) 1ug/ml final concentration to exclude dead cells
Keep sample on ice until and during analysis and sorting.
Lin+ cells are stained to be Pe-CY5+
Murine Hematopoietic Stem
Cell/Progenitor
Sort Gating Strategy
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Doublet Discrimination
PI Neg
Lin Neg
Myeloid progenitors are sorted as Lin-Sca-1-c-kit+CD34+FcgRlo
(CMP), Lin-Sca-1-c-kit+CD34+FcgRhi (GMP) and as Lin-Sca-1-ckit+CD34-FcgRlo (MEP).
HSC and CLP are isolatable as Lin-/loSca-1hic-kithiIL-7R- and Lin-Sca1loc-kitloIL-7R+ populations, respectively
Murine Bone Marrow Staining Strategy
Koichi Akashi Lab
Murine Bone Marrow
Staining Strategy
Murine Hematopoietic Stem Cell/Progenitor
Sort
Pre sort Instrument Set-UP Procedure
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Verify stable sort streams : 4 Way spread out wide
Select Parameters necessary
Run Auto Compensation program
- unstained control for PMT balance and
adjust scatter gate to include dead and live
cells
- scan each stained B220 spleen sample for
intensity of stain and uncomped
overlap adjust if necessary
Then run Auto Comp : Run PI last
Murine Hematopoietic Stem Cell/Progenitor
Sort
Pre sort Instrument Set-UP Procedure II
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Rerun single color controls through all
2 parameter combo matrix
Display using normal and then
Biexponential Display to check for
under or over compensation
Adjust compensation if required and
apply to global instrument settings
Murine Hematopoietic Stem Cell/Progenitor Sort
Pre sort Instrument Set-UP Procedure III
Compensation Verification
0% Comp
3% Comp
Murine Hematopoietic Stem Cell/Progenitor Sort
Pre sort Instrument Set-UP Procedure III
Final Pre-check
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Place tubes in sort holders
Turn on test stream
Open Deflection Plate Door
Turn on test streams : check for Arcing
Open waste drawer: Center sort streams in each tube
Close drawer/door and test streams
Place siliconized eppendorf tubes in sample collection holder
Run sample set up sort gates
Sort one or two rounds of additional sorting of same gates to
eliminate contaminating cells and doublets
Wash with 75% ethanol and saline between each round to
eliminate residual cells
Gating sequence Murine Bone Marrow
Myeloid Progenitor Sorting on
FACSAria
Murine Hematopoietic
Stem Cell/Progenitor Sort
References
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1. Kondo M, Weissman IL, Akashi K. Identification of
clonogenic common lymphoid progenitors in mouse bone
marrow. Cell. 1997;91:661-672.
2. Akashi K, Traver D, Miyamoto T, Weissman IL. A
clonogenic common myeloid progenitor that gives rise to all
myeloid lineages. Nature. 2000;404:193-197.
3. Miyamoto T, Weissman IL, Akashi K. AML1/ETOexpressing nonleukemic stem cells in acute myelogenous
leukemia with 8;21 chromosomal translocation. Proc Natl Acad
Sci U S A. 2000;97:7521-7526.
Sorting is believing
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Sorting of rare event populations allow
further determination of functional
activity
Validation of Sort via model rare event
sorting is essential
Experiment feed back aids in future
experiment design strategy
ACKNOWLEDGEMENTS
SINGAPORE
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BOSTON
Jay Dong
Melinda Leong
Gerard Chew
All of you (Course
Organizers, Attendees
and Participants) for
your interest in flow
cytometry!
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Suzan Lazo-Kallanian
Jerome Ritz
Ken Anderson
Jim Griffin
Lee Nadler
All HemNeo Flow P.I’s and
Users
All Boston BD Staff
– Paul Melanson, Michelle
LeQue
– Randy Offord, Stephanie
Ventullo
GERARD TEOH
(for being a good guy)