Flow Cytometry at the Quad

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Flow Cytometryat the Quad

• The Harvard Medical School Experience
• 1978-2008

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Flow Cytometry in the Longwood Medical Area2008

• Used ubiquitously
• Single and core flow facility format
• Self service analyzers/some self serve sorters
• New technologies Lasers/Image/High Throughput
  systems
• Biotech integration start ups/large biopharma
• Self assembling user groups BUG,BBG,BHUG
• Around the clock usage

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The Past
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1978
2008

• 34 Sorters
• too many to estimate(180)

• 3 SORTERS
• 1 ANALYZER

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In the Beginning

• Dr Albert Coons Harvard Medical School
  Pathology Department

Fluorescein Isothiocyanate FITC
1941
Immunological properties of an antibody
containing a fluorescent group. Proc.
Soc. Exp. Biol. Med. 47200
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The Coulter Principle Cells traveling in a
liquid Stream Circa 1950s
Wallace Coulter

• His interest went beyond work being done in
  hematology. In particular, he was always
  interested in expansion of knowledge in flow
  cytometry.

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Mack Fulwyler Los Alamos National
Laboratory Circa early 60s
Dr Leonard Herzenberg Dr. Lee Herzenberg
Stanford University FACS Circa late
60s early 70s
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The FACS I
Fluorescence Activated Cell Sorter
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Instrument Evolution Becton Dickinson Stream
Alignment
1978
2008
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3 SORTERS 1 ANALYZER
1978
Sam Latt M.D. PhD
Stuart Schlossman M.D.
Dr. Benacerraf Nobel Prize in Medicine 1980
Howard Shapiro M.D.
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late 70s- mid 80s

• Schlossman Tumor Immunology T Cell Human
  Monoclonal antibodies Hybridoma Technology CD4
  CD8 T cell receptor, etc
• Benacerraf Pathology Murine Immunology Noble
  Prize 1981 MHC/Ir gene response
• Latt Cytogenetics Chromosome /DNA Sorting/
  Analysis
• Shapiro (CytoKinetics) Innovator (DIY) Do It
  Yourselfer
• --------------------------------------------------
  -----
• BD Commercialized FACS from Stanford
• Ortho monoclonal Abs and instrumentation
• Coulter Sophisticated Cytometers and Monoclonal
  Antibodies

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Tumor ImmunologySidney Farber Cancer Institute
circa 1980
If you want to know where T-cells come
from Dont ask Masters and Johnson ask
Reinherz and Schlossman. from Practical
Cytometry By Howard Shapiro
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The Cutting Edge

• Magic Bullets
• Understanding of Immune regulation
• Apoptosis
• Signal Transduction
• Cell manipulation and Gene Therapy
• Small Molecule drug delivery
• Stem cell Therapy
• Cell Biology Systems

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Sorting Milestones

• Commercial Instrumentation
• Electronic Sorting for Functional assays
• Monoclonal Antibodies
• Novel biological fluorescent probes
• Development of Cyanine Dyes
• Creation of Analyzers
• High Speed Sorting MoFlo
• 4 way sorting/ auto cloning
• GFP
• Fixed Alignment Sorters
• Solid state lasers
• Diverse users and applications

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The 80s
The 90s
The Decades of Flow

• Era of monoclonal antibody integration
• Instrumentation sophistication
• Application expansion
• Technology integration
• Flow core creation
• Clinical standardization

• Infrastructure Expansion
• Molecular Biology GFP
• Local Cytometry Users Groups
• Wide Spread Usage

The New Millennium

• Drug Discovery Cell Cycle
• High Throughput
• Kit simplification
• Stem cell Extensive Multicolor
• Fluorescent Protein Sorting
• Instrument standardization

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PRESENT Future
Hematologic Neoplasia Flow Cytometry Core Facility
http//research.dfci.harvard.edu/flowlab/
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Simultaneous sorting of 4 distinct progenitor
populations using the FACSAria. Sorting of Murine
bone marrow cells at 15,000-20,000 cells/sec
utilizing 6 color fluorescence (FITC, PE, PI,
PE-Cy5, APC, and APC-CY7) into four populations
simultaneously. Cells are first gated to be PI -
and Lin-. The HSC population is then sorted alone
and the progenitor region is sub sorted into
three separate populations. MEP, GMP, and CMP
Multiparameter Flow Cytometry assays of Cre and
Cre- Hematopoietic Stem Cells(HSC).Reactive
Oxygen Species (ROS) analysis using 5 µM DCF-DA
, cell cycle analysis using Hoechst 33342 and
Apoptosis analysis using Annexin V and 7AAD
Gary Gilliland, Zuzana Tothova, Sorter
FoxOs Are Critical Mediators of Hematopoietic
Stem Cell Resistance to Physiologic Oxidative
Stress CELL Jan 26 2007. P325 Zuzana Tothova
et al
Murine Stem Cell Polychromatic Flow Cytometry
Assay Integration
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GFPDsRed/Hoechst 33342Viable Cell CycleAssay
IntegrationSelf Service Sorting
Sherry Yen Steve Elledge Lab
Hoechst Blue450BP filter GFPFITC 530
filter DsRedPE 575 filter
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20mW Ultraviolet Flow Cytometry Applications
Six Color Calcium flux of T cell subsets using
Indo-1 and 20mW UV excitation. Purified
mononuclear cells were labeled with the following
antibodies CD3 Pacific Blue/CD45RAFITC/CD45RO
PE/CD4 PE-CY7 and loaded with INDO1-AM. Cells
were run on UV SORP FACSAria for 30 seconds
removed and 10ug of Goat Anti-Mouse IgG1 was
added to cross link antibodies and observe
calcium flux in various subpopulations over five
minutes. A separate sample was treated with
ionomycin as a positive control.
Minus Verapamil
Verapamil
H O B L U E
HOECHST RED
Sorting of Side Population (SP) of Cell line
using 20mW UV laser on SORP FACSAria.
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20mW Ultraviolet Flow Cytometry Applications
5 color analysis and sorting of murine bone
marrow using DAPI as a viability probe 5 color
fluorescence (c-kit APC, sca-1 PE, CD34 FITC,
LIN-, and DAPI) of Murine Bone Marrow using UV
excited DAPI as a viability dye.
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High Throughput circa 1979 aka Micro-Sample
Delivery System
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High
Throughput 384 well Plate acquisition and
analysis on FACSCanto II 100ul of Gr-1 PE labeled
mouse bone marrow was manually loaded into each
well of a 384 well microtiter plate and processed
on a FACSCanto II flow cytometer equipped with
the High Throughput Sampler Option (HTS). 3ul of
sample per well was acquired and recorded for
analysis. Total acquisition time to run one 384
well plate was approximately 50-60
minutes. FACSDiva 6.1.1. Batch analysis was
performed on the 384 samples and data was stored
graphically in Adobe PDF format and statistics on
each well were batched together as a single
file and exported to a Microsoft Excel
spreadsheet. Excel line charts were generated
that displayed results from the entire 384 well
plate that yielded information pertaining to
number of events per well processed, percent G-1
reactivity/well, and median fluorescence
intensity(MFI) of GR1-PE/well.
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Dealing with the Data FACSCanto II Yeast
Study CFPYFP HTS Application
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Bottom Line Data Display
1. Set-Up
2. Fine tune Acquisition and analysis
3. Display in Understandable format
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Rapid Data OutputBatch Statistic AnalysisExcel
spreadsheet
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Works in Progress

• High Throughput Flow Cytometry
• Image Integration
• Front and Back end Software

Flow Cytometry Where the Stream meets the Beam
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http//weinstock.hccdev.org/
Zinc Finger Nucleases
Load Robotically
6ooo cells/well
High Content screening microscopy
384 well image plate
DNA Repair
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The Challenge
Chemical Screen 20,000 compounds
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Post Image Analysis By HTS Flow
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HyperCyt meets Canto
Peristaltic pump delivery
96 well plate run 2.5 minutes 384 well plate
run 10 minutes
portable
Heat map for Hits
Minor instrument modifications
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Randomly added GFP cells to a plate of
mostly GFP Negative Cells
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Flow and Image Cytometry Integration Sorting of
minor populations staining via
immunohistochemistry
Sort Cytospin Immunohistochemistry Fluor
escence Micrscopy
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Chicken Red Blood Cells An Ancient Flow
Cytometry Standard
Fluorescence Microscopy
Flow Cytometry
Amnis Image Stream
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DNA Repair Foci Formation
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561 Yellow-Green Laser Improved mCherry Detection
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561nm Yellow Green Laser Improved separation
of PE and PE Tandem probes
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Instrument Development 1970s Timeline - 2008
ORTHO(JJ)
COULTER
CYTOMATION
CYTOPEIA
BD
others!
Credits J. Paul Robinson/Purdue
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others!
Instrument Development (contd)Timeline NOW
others!
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Then and NowFACS Applications
1978
2008

• Cell surface Phenotype
• DNA Analysis
• Chromosome sorting
• Single color sorting of lymphoid subpopulations
• Exotic applications

Sorting Applications

• Polychromatic 4 way sorting of minor
  subpopulations
• Viable cell cycle sorting
• GFP sorting
• PCR single cell sorting

A N A L O G
D I G I T A L

• Analysis Applications
• Patient Phenotype Profiling
• Apoptosis
• Cell Cycle
• GFP
• ROS
• Polychromatic Immunofluorescence
• Proliferation
• mCherry
• DNA repair
• Rare event analysis
• High throughput screening
• Calcium flux kinetics

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Daleys Three Laws of Flow

• Balance
• Pattern Recognition
• Intelligence

The Sky is the Limit.