Recent Advances in Multicolor Flow Cytometry Applications

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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_at_dfci.harvard.edu
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Recent Advances and Applications at HemNeo Flow
http//research.dfci.harvard.edu/flowlab

• 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
• GFPDsRed 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
• Rare event Sorting Instrument considerations ,
  Haploid sperm cells, Human Tetramer Sorting,
  Single cell PCR cloning
• Addition of FACSCanto II switching to plates
  and analyzing by batch

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FACSAria Special Order Research Platform
(SORP)UV Laser 20mW 355nm
UV PMT TRIGON
SORP OPEN OPTICAL BENCH
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UV Applications

• 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)

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FACSAria SORP UV Laser Upgrade January 2007
GFPDsRed/Hoechst 33342Viable Cell CycleAssay
Integration
Hoechst Blue450BP filter GFPFITC 530
filter DsRedPE 575 filter
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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

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FACSAria UV ApplicationsCalcium Flux Kinetic
Assays
Ionomycin
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Calcium Flux

• Method to get Gap in Time vs. ratio Histogram (
  Lift Cover/reset time stop/append file/close
  cover after 5 seconds)

Indo-Violet405nm BP Filter Indo-Blue530nm BP
Filter Dichroic505nm LP
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Calcium Flux on T cell subpopulations
FACSAria SORP 20Mw 355nm UV Laser
10ug GAM IgG1
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Side Population (SP) Analysis and
SortingBackground History

• 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

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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
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Side Populations (SP)by Flow

• Stringent Protocol Adherence
• Instrument Parameters Critical
• Tight C.V.s (co-efficient of Variation)
• Optimize Optical Filter selection
• Instrument Alignment Conditions

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UV Bead StandardFluoresbrite BB Carboxylate
Microspheres 4.5µmPolysciences
LOG
LINEAR
Hoechst Blue PMT 225 Volts
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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
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Side PopulationsInstrument Parameters

• 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)

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SP Labeling procedures

• Verify technique by using same strain and EXACT
  rigorous conditions
• Found in above mentioned protocol
• Maintain incubation precisely at 37 centigrade
• 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

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SP Labeling procedures

• 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

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Side Population enumeration using Hoechst 33342
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SP PhenotypeMurine Bone Marrow
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Ho33342 dye uptake kinetics - incubation time and
concentration titration important
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Violet Excitation SP HO33342 BM
Verapamil
0.4
0.3
PMT blue 550 PMT Red 944
Spectral Viewer
William Telford N.I.H.
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Ultraviolet 20mW 355nm SP HO33342 BM
Verapamil
HO BLUE (450)
HO RED (670)
PMT blue 316 PMT Red 584
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Other Interesting Developments
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Multiple Gating strategies help identify low
frequency functional subpopulations
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Multicolor(5) Minor populations Four way
sorting Treg Story
CD45ra FITC CD127 PE CD25 PE-CY5 CD4 PE-CY7 CD3
PAC BLUE
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Treg Post Sort Reanalysis
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POST SORT REANALYSIS HUMAN PBL CD3CD4 CD45RA
CD45RA-
Treg/Teffector/ --
98.4
99.4
97.1
99.9
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FoxP3 Expression in CD4 Treg CD45RA
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Fluorescence minus 1
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Rare Events The Needle in the Haystack Story
Burn the Haystack
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Rare Events and Flow Cytometry

• 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

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GFP 0.7-? 99 20,000 sec
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Rare Event Tetramer Sorting 0.007
bead auto compensation
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Tetramers Ag Specific T cellsSmall events
bright labelheavy gating
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Rare Events and Flow CytometryPractical
Considerations

• 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?

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Rare Events and Flow CytometryInstrument
considerations

• 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

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Rare Events and Flow CytometryExperimental
Design Strategy

• 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

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4 way Bead sort to check Instrument Accuracy
Protocol

• Beads needed
• 1 Accudrop cat345249
• 2 Calbrite APC cat 340487
• 3 Sphero Rainbow Fluorescent particles 
  3.0-3.4um(mid range FL1) cat 556298
• 4   "           "                "              
  "            "          ( brighter?)     cat
  556291
•  
•  
• 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
•  
• Second Add a few drops of each bead to separate
  tube add 1xpbs(200ul)    and run/
  record 5,000-10,000events
•  
• Third     Adjust  APC Cy7 down to 385 volts when
  run Accudrop  to get beads on scale. Use
  Biexponential display option
•  
• 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)
•  
• 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.   

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Simple Four Way bead Sort
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4 way bead sort Spiked with Fixed Murine Spleen
cells
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Isolation of minor subpopulations G. Daley
Lab (Melissa H)
Mouse embryonic stem cells/ Sperm cell
Precursors Nature December 10, 2003
Picture credit Niels Geijsen
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Rare Event Gating Strategy Pre Sperm
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RT-PCR Single Cell Cloning

• One is one
• The ultimate rare event

Sophia Adamia Ken Anderson Lab DFCI
Thermocycler Plate Bottom cut off to fit in ACDU
unit
Adjust stream not plate
Check to see if Green beads on bottom Of vial not
sidewall 10uL lysis Buffer
Flash drive cover Used as wedge
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RT-PCR by Flow Cytometry
Waldenstroms macroglobulinemia CD19 (B cells )
from patient bone marrow aspirate

1. PCR Reaction Thermocycler 2. Capillary
electrophoresis on ABI DNA Genetic Analyzer
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DNA Fragment Analysis
Capillary electrophoresis
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Expression of SIVA splice variants in healthy
donor BM CD19B cells
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Murine Hematopoietic Stem Cell/Progenitor
SortKoichi Akashi LabDFCI
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

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Murine Hematopoietic Stem Cell/Progenitor
SortStaining strategy
HSCs and CLPs FITC-Sca-1
bioinylated IL-7RPE 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
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Murine Hematopoietic Stem Cell/ProgenitorSort
Gating Strategy

• Doublet Discrimination
• PI Neg
• Lin Neg
• Myeloid progenitors are sorted as
  Lin-Sca-1-c-kitCD34FcgRlo (CMP),
  Lin-Sca-1-c-kitCD34FcgRhi (GMP) and as
  Lin-Sca-1-c-kitCD34-FcgRlo (MEP).
• HSC and CLP are isolatable as Lin-/loSca-1hic-kith
  iIL-7R- and Lin-Sca-1loc-kitloIL-7R populations,
  respectively

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Murine Bone Marrow Staining Strategy
Koichi Akashi Lab
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Murine Bone Marrow Staining Strategy
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Murine Hematopoietic Stem Cell/Progenitor
SortPre sort Instrument Set-UP Procedure

• 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
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Murine Hematopoietic Stem Cell/Progenitor
SortPre sort Instrument Set-UP Procedure II

• 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

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Murine Hematopoietic Stem Cell/Progenitor
SortPre sort Instrument Set-UP Procedure
IIICompensation Verification
0 Comp 3 Comp
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Murine Hematopoietic Stem Cell/Progenitor
SortPre sort Instrument Set-UP Procedure
IIIFinal Pre-check

• 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

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Gating sequence Murine Bone Marrow
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Murine Hematopoietic Stem Cell/Progenitor Sort
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References

• 1. Kondo M, Weissman IL, Akashi K. Identification
  of clonogenic common lymphoid progenitors in
  mouse bone marrow. Cell. 199791661-672.
• 2. Akashi K, Traver D, Miyamoto T, Weissman IL. A
  clonogenic common myeloid progenitor that gives
  rise to all myeloid lineages. Nature.
  2000404193-197.
• 3. Miyamoto T, Weissman IL, Akashi K.
  AML1/ETO-expressing nonleukemic stem cells in
  acute myelogenous leukemia with 821 chromosomal
  translocation. Proc Natl Acad Sci U S A.
  2000977521-7526.

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Sorting is believing

• 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

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ACKNOWLEDGEMENTS
BOSTON
SINGAPORE

• Jay Dong
• Melinda Leong
• Gerard Chew
• All of you (Course Organizers, Attendees and
  Participants) for your interest in flow
  cytometry!

• Suzan Lazo-Kallanian
• Jerome Ritz
• Ken Anderson
• Jim Griffin
• Lee Nadler
• All HemNeo Flow P.Is and Users
• All Boston BD Staff
• Paul Melanson, Michelle LeQue
• Randy Offord, Stephanie Ventullo

GERARD TEOH (for being a good guy)