flow cytometry applications for isolating and … flow cytometry applications for isolating and...
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![Page 1: Flow Cytometry Applications for Isolating and … Flow Cytometry Applications for Isolating and Analyzing Complex Heterogeneous Stem Cell Cultures Nil Emre, PhD Manager, Stem Cell](https://reader031.vdocument.in/reader031/viewer/2022021821/5af7a7787f8b9a7444910d35/html5/thumbnails/1.jpg)
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Flow Cytometry Applications for Isolating and
Analyzing Complex Heterogeneous Stem Cell Cultures
Nil Emre, PhD
Manager, Stem Cell Research and Development
BD Biosciences
23-15381-00
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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• Background
• Immunophenotyping Screens
• Improved Sorting of Pluripotent Stem Cells
• Enrichment for hiPSCs during Reprogramming
• Monitoring Lineage Specification Using Multiparameter Flow Cytometry
• MSC Characterization Using Flow Cytometry
Overview
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Applications of stem cell research:
• Cell therapy
• In vitro disease models
• Basic research
Heterogeneity of cell cultures is a major challenge.
The field is in need of:
• Defined cell-surface signatures for pluripotent stem cells and their derivatives
• Robust methods for cell sorting
• Quantitative analysis tools for heterogeneous cell cultures
The BD Biosciences Stem Cell Research group in La Jolla, CA is addressing
these hurdles:
• Developing antibody-based reagents and applications for flow cytometry and
bioimaging technology platforms
• Working with key opinion leaders in technology development
Applications and Challenges in Stem Cell Research
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Immunophenotyping Screens for Identifying Cell-
Surface Signatures of Human Embryonic Stem Cell
(hESC) and Human Induced Pluripotent Stem Cell
(hiPSC) Derived NSCs, Neurons, and Glia by Flow
Cytometry
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Can we use our portfolio of antibodies to cell-
surface markers to identify signatures of stem cells
and their derivatives outside hematopoiesis?
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Enabling researchers to immunophenotype cell populations by flow cytometry or
immunofluorescence microscopy using BD’s portfolio of monoclonal antibodies
Product Contents Size
BD Lyoplate™ Human
Cell Surface Marker Screening Panel
Cat. No. 560747
• 242 CD markers*
• Isotype controls
• Alexa Fluor 647 second step
5 tests
BD Lyoplate™ Mouse
Cell Surface Marker Screening Panel
Cat. No. 562208
• 176 CD markers
• Isotype controls
• Biotin second step
• Alexa Fluor 647 streptavidin
third step
5 tests
*CD and other cell-surface molecules. One marker per well.
• Accelerate the discovery of unique cell-surface “signatures”
• A unique, cost-effective alternative to screening using
hundreds of single-vial reagents
Alexa Fluor® is a registered trademark of Molecular Probes, Inc. The Alexa Fluor® dye antibody conjugates in this product are sold under
license from Molecular Probes, Inc., for research use only, excluding use in combination with microarrays, or as analyte specific reagents.
BD Lyoplate™ Screening Panels
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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August 2011, Volume 6, Issue 8
October 2011, Volume 29, Issue 11
Stem Cell Research Screening Applications
December 2012, Volume 7, Issue 12
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March 2011, Volume 6, Issue 3
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The field needs:
– Robust standardized methods for
isolating NSC to eliminate batch to batch
variability
– Robust standardized methods for
isolating terminally differentiated
neurons
– Identification of transplantable cell types
that do not cause tumors
Sort
Sort
Characterization of cell-surface
signatures enables:
– Identification of subpopulations
• Transplantation
– Isolation of pure populations
• Arrays
• Biochemistry
• In vitro assays and models
– Development of quality control
assays for cell preparations
• Purity assessment
• Contaminant identification
Neural Induction of Pluripotent Stem Cells
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CD marker “D”
hESCs
NSC Contaminants
EB Rosette+
EB Rosette–
NSCs
CD marker “C”
CD marker “B”
CD marker “A”
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
54.9 45.1
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
2.88 97.1
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
1.3 98.7
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
2.1 97.9
10 0
10 1
10 2
10 3
10 4
APC-A
0
20
40
60
80
100
% o
f M
ax
83.3 16.7
10 0
10 1
10 2
10 3
10 4
APC-A
0
20
40
60
80
100
% o
f M
ax
1.27 98.7
10 0
10 1
10 2
10 3
10 4
APC-A
0
20
40
60
80
100
% o
f M
ax
0.7 99.3
10 0
10 1
10 2
10 3
10 4
APC-A
0
20
40
60
80
100
% o
f M
ax
14.8 85.2
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
22.5 77.5
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
4.87 95.1
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
18.1 81.9
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
99.8 0.22
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
11.2 88.8
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
1.47 98.5
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
1.71 98.3
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
68.8 31.2
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
99.4 0.57
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
87.2 12.8
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
98.5 1.55
0 10 2
10 3
10 4
10 5
APC-A
0
20
40
60
80
100
% o
f M
ax
65 35
Cell Surface Marker Screen: Flow Cytometry
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% Positive Plate 1
MFI Plate 1
MFI Plate 2
bdbiosciences.com/resources/stemcell/
% Positive Plate 2
Analysis
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Sox1 Sox2 Nestin
99.1% 98.5% 99.4%
CD184+/CD44–/CD271–/CD24+ Define a Cell-
Surface Signature for the Isolation of NSCs
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H9 NSCs differentiated for 3 weeks
Data courtesy of Mike Hefferan and Martin Marsala, UCSD
HUES-9 NSCs 8 weeks post-
engraftment in rat spinal cord
Sorted NSCs Differentiate to Mixed Cultures of
Neurons and Glia
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Glia:
CD184+/CD44+
Tuj1
Nestin
GFA
P D
NA
Neurons:
CD184-/CD44-/CD15lo/CD24+
Isolation of Neurons and Glia by Sorting
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CD184+/CD271-/CD44-/CD24+
CD184-/CD44-/CD15LOW/CD24+ CD184+/CD44+
• Defined cell-surface signatures for NSCs, neurons, and glia
• Signatures enable:
Standardized and robust isolation of hESC-derived neural stem cells
Downstream applications requiring consistent or pure cell populations
• The immunophenotyping principle can be applied in other stem cell fields.
Summary
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bdbiosciences.com Alzheimer’s disease biochemical marker comparative analysis
Reprogramming
Fibroblasts
Directed neuronal differentiation
and sorting purification
Purified Neurons
iPSCs
Nature January 2012, Volume 482, Issue 7384
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Sort:
CD184+
CD24+
CD44-
CD271-
Sort:
CD184-
CD44-
Sort:
CD184+
Glia
Neurons
CD15lo
CD24+
CD44+
Neurons
Neural induction
culture
Sort?
Neurons, Glia
and other cell types
Cell-Surface Signatures for the Isolation
of Neurons
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CD200
CD340 HLA-A,B,C CD49f
FSC
DC
X
Sox1
13.7%
20.3% 46.8%
19.3%
SS
C
Neurons
NSCs+
Stain with BD Lyoplate™
Human Cell Surface Marker
Screening Panel
Stain with Sox1, Sox2, Pax6,
and DCX
6-Week Neural Induction
Culture
Dis
asso
cia
te
Cu
ltu
res
Fix
Perm
Analyze cell-surface marker
expression of populations
defined by intracellular
markers
HT
S F
low
Cyto
metr
y
Identification of Cell-Surface Marker Signatures
of Neurons
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FSC-A
SS
C-A
84.7%
CD200
HL
A-A
,B,C
NSC+ 67.9%
Neurons 25.8%
CD
20
0m
id
HL
A-A
,B,C
+
91.5%
1.0%
NSC+
Neurons
So
x2
DCX
Tuj1Ki-67NestinDNA
Pre
-So
rt
61.0%
22.4%
NSC+
Neurons
So
x2
DCX
CD
20
0h
i
HL
A-A
,B,C
– 4.4%
82.0%
So
x2
NSC+
Neurons
DCX
Isolation of Neurons Using CD200 and HLA-A,B,C
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Sort:
CD184+
CD24+
CD44-
CD271-
Sort:
CD184-
CD44-
Sort:
CD184+
Glia
Neurons
CD15lo
CD24+
CD44+
Neurons
Neural induction
culture
Sort:
HLA-A,B,C–
CD200hi
Neurons, Glia
and other cell types
Without IC flow:
Screened 6 different cell cultures
With IC flow:
Screened 1 cell culture
Cell-Surface Signatures for the Isolation
of Neurons
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Improved Sorting of Pluripotent Stem Cells
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• Are sorted hESCs viable?
– Fong CY, Peh GS, Gauthaman K, Bongso A. Separation of SSEA-4 and TRA-1-68 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Stem Cell Rev. 2009;5:72-80.
– Nicholas CR, Gaur M, Wang S, Pera RA, Leavitt AD. A method for single-cell sorting and expansion of genetically modified human embryonic stem cells. Stem Cells Dev. 2007;16:109-117.
– Sidhu KS, Tuch BE. Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization. Stem Cells Dev. 2006;15:61-69.
• Do sorted cells still express markers of pluripotency?
• Are sorted cells capable of further differentiation?
• No commercial, standardized methods for sorting hESCs by flow cytometry.
Sorting and Analysis of Pluripotent Stem Cells
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Sorting and Analysis of Pluripotent Stem Cells
• The ROCK inhibitor, Y-27632, enhances survival of hESCs after single-cell dissociation.
– Narumiya S, Ishizaki T, Uehata M. Use and properties of Rock-specific inhibitor Y-27632. Methods Enzymol. 2000;325:273-284.
– Watanabe K, Ueno M, Kamiya D, et al. A Rock inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007;681-686.
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Sorting Based on Markers for Pluripotency and
Differentiation
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ROCK Inhibitor Y-27632 Increases Cell Recovery
Post Sort
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Sorted hESCs Retain Pluripotency
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• Sorted hESCs are viable and recoverable.
• Recovery of cells is improved by the addition of Y-27632.
• Sorted cells express markers of pluripotency, are able to differentiate, and can maintain a normal karyotype.
• Standardized method for sorting hESCs by flow cytometry.
The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-
activated cell sorting with multiple cell surface markers. Nil Emre, Jason G. Vidal, Jeanne Elia, Eric D.
O'Connor, Rosanto I. Paramban, Michael P. Hefferan, Roman Navarro, Danielle S. Goldberg, Nissi M. Varki,
Martin Marsala, and Christian T. Carson. PLoS One, 2010.
Sorting and Analysis of Pluripotent Stem Cells
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Enrichment for hiPSCs During Reprogramming
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Flow Cytometry and hiPSCs
BD Stemflow™ Human Induced Pluripotent Stem Cell Analysis and Sorting Kit
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SS
EA
-4
Ale
xa
Flu
or®
64
7
FSC-A
SS
C-A
CD
13
Pe
rCP
-Cy5
.5
FSC-A
Cells at Day 21 Post-Transduction
Cells at Day 35 Post-Transduction
SS
EA
-4
Ale
xa
Flu
or®
64
7
SS
C-A
CD
13
Pe
rCP
-Cy5
.5
TRA-1-60 PE FSC-A FSC-A
TRA-1-60 PE
18%
20%
19%
10%
Data courtesy of Peter Flynn and Bob Valamehr, Fate Therapeutics
Reprogramming Time Course
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SS
EA
-4
Ale
xa
Flu
or®
64
7
SS
C-A
CD
13
Pe
rCP
-Cy5
.5
TRA-1-60 PE FSC-A FSC-A
Day 6 Post-Sort Day 11 Post-Sort
Data courtesy of David Kahler, New York Stem Cell Foundation
99%
83%
Sorting Established hiPSC Lines
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BD Pharmingen™ Cell Sorting Preparation
Buffer Set
• Preparation and maintenance of single-cell suspensions for sorting and analysis
• Cell types tested for sorting applications: – Human bone marrow
– T cells
– Regulatory T cells (Tregs)
– hESCS
– NSCs
– Neurons
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4.2%
5.3%
Complete Media
Sorting Buffer
End of Sort Viability Comparison: hESCs
PI
SS
C-A
5.3%
4.2%
SS
C-A
PI
SS
C-A
FSC-A
SS
C-A
FSC-A
4.2%
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Post-Sort Analysis: hESCs
SS
C-A
FSC-A
Nan
og
0.2%
Cells Alone Sorting Buffer
98.1% 96.7%
Complete Media
Oct4A
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Monitoring Neural Ectoderm and Endoderm
Differentiation from hESCs using Multiparameter Flow
Cytometry
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b-III tubulin Nestin DNA
Differentiation of hESC-derived NSCs to neurons and glia
BD Stemflow™ Neural Lineage Analysis Kit
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Specificity Format Molecule Neural Cell Population
Identified
CD44 FITC H-CAM Glial precursors, astrocytes
Ki-67 Alexa Fluor® 488 Proliferation marker All proliferating cell types
Doublecortin PE Neurofilament Immature neurons
Sox2 PerCP-Cy™5.5 Transcription Factor NSCs, glial precursors
Sox1 PerCP-Cy5.5 Transcription Factor NSCs, glial precursors
GFAP Alexa Fluor® 647 Filament Mature astrocytes
Nestin Alexa Fluor® 647 Filament NSCs, glial precursors
Buffers Included
BD Cytofix™ fixation buffer
BD Phosflow™ perm buffer III
BD Stemflow Neural Lineage Analysis Kit
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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Differentiation of hESC-derived NSCs to neurons and glia
DC
X
Day 7 Day 14 Day 21 Day 27 Day 0
6.5%
90.5%
7.0%
80.6%
17.3%
73.7%
35.4%
54.6%
40.0%
49.8%
Nestin
DC
X
Nestin
CD
44
3.5% 5.3% 21.2% 35.4%
Neuron
NSC & glia
glia
BD Stemflow Neural Lineage Analysis Kit
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Endodermal Differentiation from hESCs
Nanog Sox17 FoxA2 DAPI Nanog Oct-4 Sox2 DAPI
Goal: To develop quantitative flow cytometric assays to monitor endodermal
differentiation
3 Days
Low FBS, Activin
(D’Amour et al, 2005)
hESCs Definitive Endoderm
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Panel Components
Specificity Clone Isotype Format
Pax-6 O30-1330 mIgG1, κ Alexa Fluor® 488
PDX-1 658A5.1.37 mIgG1, κ PE
FoxA2 N17-280 mIgG1, κ PE
Sox2 O30-678 mIgG1, κ PerCP-Cy5.5
Sox17 P7-969 mIgG2a, κ PerCP-Cy5.5
Nanog N31-355 mIgG1, κ Alexa Fluor® 647
BD Stemflow™ Definitive and Pancreatic Endoderm
Analysis Kit
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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Mark Moorman, ViaCyte, Inc.
FoxA2 Pdx1 Nanog
So
x1
7
0.2%
0%
99.2%
0.5%
1.2%
96.8%
0.2%
95.6%
99%
0%
0%
0%
1.7%
97.5%
95.9%
1%
0.8%
4%
Day 0
Day 2
Day 12
CyT49 hESCs
Definitive
endoderm
Pancreatic
endoderm hESC
Monitoring Endodermal Differentiation Using Flow
Cytometry
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MSC Characterization using Flow Cytometry
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• MSCs must be plastic adherent.
• MSCs must express CD73, CD105, and CD90 and lack
expression of CD45, CD34, CD14, CD11b, CD79a, and
CD19.
• MSCs must differentiate into osteoblasts, adipocytes, and
chondrocytes in vitro.
MSC Definition
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MSC Variability
Intra-population heterogeneity:
- Early vs late passage in culture
- Normoxia vs hypoxia
- Serum vs serum free
- Subsets of MSCs with distinct functions and potential
Source:
- Bone marrow
- Adipose tissue
- Umbilical cord
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MSC Heterogeneity
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BD Stemflow™ Human MSC Analysis Kit
0 102
103
104
105
Re
lative
Ce
ll N
um
be
r
100
0 102
103
104
105
Re
lative
Ce
ll N
um
be
r
100
0 102
103
104
105
Re
lative
Ce
ll N
um
be
r
100
0 102
103
104
105
Re
lative
Ce
ll N
um
be
r
0.655
Lin PE CD105 PerCP-Cy5.5 CD73 APC CD90 FITC
GD2 GD2
SS
EA
-4
CD
14
6
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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Heterogeneity of stem cell cultures is a major challenge
Potential solution:
• Defined cell-surface signatures for stem cells and their derivatives
– BD Lyoplate™ Human Cell Surface Marker Screening Panel
– BD Lyoplate™ Mouse Cell Surface Marker Screening Panel
• Robust methods for cell sorting
– BD Stemflow™ Neural Sorting Kit
– BD Stemflow™ Pluripotent Stem Cell Sorting and Analysis Kit
– BD Stemflow™ Human Induced Pluripotent Stem Cell Analysis and Sorting Kit
– BD Pharmingen™ Lineage Cocktail 4 (lin 4)
– ROCK Inhibitor (Y-27632)
– BD Pharmingen™ Cell Sorting Preparation Buffer Set
• Quantitative analysis tools for heterogeneous cell cultures
– BD Stemflow™ Definitive and Pancreatic Endoderm Analysis Kit
– BD Stemflow™ Neural Lineage Analysis Kit
– BD Stemflow™ Human MSC Analysis Kit
– BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit
– BD Stemflow™ Human Pluripotent Stem Cell Transcription Factor Analysis Kit
– BD Stemflow™ Mouse Pluripotent Stem Cell Transcription Factor Analysis Kit
The Devil is in the Details: Heterogeneity
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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BD Research and
Development: Christian Carson
Mirko Corselli
Ravi Hingorani
Amy Hsieh
Jody Martin
Erika O’Donnell
Jurg Rohrer
Jason Vidal
Lissette Wilensky
UCSD Larry Goldstein
Martin Marsala
Eric D. O’Connor
ViaCyte, Inc. Mark Moorman
Fate Therapeutics Peter Flynn
Bob Valamehr
New York Stem Cell Foundation David Kahler
Dorota Moroziewicz
Matthew Zimmer
BD Colleagues Bob Balderas
Trent Colville
Andrea Nguyen
Dennis Sasaki
Acknowledgments
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If you have further questions:
Contact your US Reagent Sales Rep
or e-mail: [email protected]
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
Cy™ is a trademark of GE Healthcare.
BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2013 BD