Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir...
-
Upload
lynne-stokes -
Category
Documents
-
view
217 -
download
0
Transcript of Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir...
![Page 1: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/1.jpg)
Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection
Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky
![Page 2: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/2.jpg)
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu153
Ho
16
5
Flow Cytometer with (elemental) Mass Spectrometer Detection
•advantage
•metal-labeled tags
•bulk (average) assay
•cytometry (individual cell) assay
•bead assay
NN
O
O O
O
Au
![Page 3: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/3.jpg)
Fluorophores: signal overlap limits multiplex capability
0
10
20
30
40
50
60
70
80
90
100
380
403
426
449
472
495
518
541
564
587
610
633
656
679
702
725
748
771
794
wavelength
inte
nsity
![Page 4: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/4.jpg)
Fluorophores: dynamic range challenge
0
20
40
60
80
100
120
140
380
403
426
449
472
495
518
541
564
587
610
633
656
679
702
725
748
771
794
wavelength
inte
nsity
1.0
5.0
0.5
0.8
2.0
0.4
0.3
0.6
![Page 5: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/5.jpg)
the ideal reporter tagnon-degradablenon-reactivenon-interferingmany “colors”completely independent detection channelsquantitative …
a close approximation:
Ch
10
Ch
20
Ch
30
Ch
40
Ch
50
Ch
60
Log
(sig
nal)
stable isotope metal atomsmeasured by mass spectrometry
10-6 abundance sensitivity
dynamic range of 108
![Page 6: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/6.jpg)
ICP-MS detection of element-tagged cells
sample
auxiliary gas
plasma gas
~ 7500 K~ 1015 e-/cm3
sun’s surface
element labeledreagents
rf energy in
![Page 7: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/7.jpg)
Potentially suitable elements and (unique mass) stable isotopes
24 elements: 67 isotopes
La1
Hf5
Re2
Ru6
Rh1
Ir2
Pd4
Pt4
Ag2
Au1
In2
Ce1
Pr1
Nd5
Sm6
Eu2
Gd5
Tb1
Dy4
Ho1
Er4
Tm1
Yb5
Lu1
13 lanthanides: 37 unique isotopes
![Page 8: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/8.jpg)
polymer produced in collaboration with Prof. Mitch Winnik, Dep’t Chemistry, U of T
SC(=S)Ph
chelator produced in collaboration with Prof. Mark Nitz, Dep’t Chemistry, U of T
chelator
SH
NN
O
O O
O
isotope tag
linker and antibody
NN
O
O O
O
synthesis and use of metal-labeled tags
![Page 9: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/9.jpg)
Antibody labeling protocol
reduce –S-S- groups
add Ln3+
ions
N
O
O
NOO
*
R
n
metal-tagged antibody
Ln(III)
ligands for Ln3+
attach polymer
S N
O
O
R N
O
O
*
n
antibody
reduce –S-S- groups
add Ln3+
ions
N
O
O
NOO
*
R
n
N
O
O
NOO
*
R
n
metal-tagged antibody
Ln(III)
ligands for Ln3+
attach polymer
S N
O
O
R N
O
O
*
n
SS N
O
O
R N
O
O
*
n
N
O
O
R N
O
O
*
n
antibody
![Page 10: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/10.jpg)
Primary anti-CD33 antibodies labeled with lab. tag-Eu and reacted with MBA-4 cells
comparison tagging reagents
MAXPAR™ (primary tags)
commercial (secondary tags)
blank (commercial 20 tags)
![Page 11: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/11.jpg)
Time /s0 100 200 300 400 500
Rh
Sig
nal /
Vol
ts
0
1
2
3
Rh+ Au+ cell 1
Rh+ Au+ cell 2
Rh+ Au+ cell 3
Rh+ Au+ cell 4
Rh+ Au+ cell 5
average of 4Rh- Au+ cells
Time /s0 100 200 300 400 500
Au
Sig
nal /
Vol
ts
0.0
0.2
0.4
Rh+ Au+ cell 6
Rh+ Au+ cell 7
Rh+ Au+ cell 8
Buffer after Au cells
a bCellular Enumeration Using Rh DNA Intercalator
[Rh(phi)2bpy]3+
![Page 12: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/12.jpg)
Analytical Protocol
triplicate tubes
live cells
CD45-Eu
30 min
1) wash PBS
2) centrifuge
3) fix 3.7% formaldehyde
4) stain metal-containing DNA intercalator
For bulk (solution) analysis:5) digest 37% HCl6) add 1.00 ppb Ir
Analyze by conventional ICP-MS
Normalize signals to Ir and Rh
For cytometric analysis:5) individual cell injection
Analyze by fast ICP-MS
Normalize signals Rh
![Page 13: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/13.jpg)
KG1anormalized response (Ir, Rh, bkgd subtracted)10-1100101102mixCD33CD34CD38CD45CD54
CD33 PrCD34 TbCD38 HoCD45 EuCD54 Tm
THP-1normalized response (Ir, Rh, bkgd subtracted)10-1100101102mixCD33CD34CD38CD45CD54
CD33 PrCD34 TbCD38 HoCD45 EuCD54 Tm
A
B
.tagged antibodies
CD54-TmCD45-EuCD38-Ho (THP-1 higher expression)CD34-Tb (THP-1 lower expression)CD33-Pr (KG-1a low expression)
Individual and 5-plex bulk assaysKG1a
THP-1
KG1a, a primitive progenitor cell, shows 500x lower expression of
CD33 than THP-1, a more differentiated cell type
Angew. Chem. Int. Ed., 46, 1–5 (2007)
![Page 14: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/14.jpg)
x400 20m
Cell Differentiation with (PMA, 72h)
PMA, 4α-Phorbol 12-Myristate 13-Acetate
10 surface markers + Rh intercalator for cell number normalization
x400 20m
DMSO 50 ng/ml PMA
THP-1
KG1a
![Page 15: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/15.jpg)
147Sm145Nd
142Nd
174Yb
146Nd
139La
170Er
141Pr
165Ho
151Eu144Nd
176Yb
169Tm
164Dy
159Tb
171Yb
152Sm
153Eu
166Er
156Gd
CD19
CD3
CD64
CD15
CD20HLA-DR
CD14
CD4
CD123
CD117
CD7
CD56
CD33
CD38
CD44CD13
CD49d
CD34
CD36
CD45
10 0 10 1 10 2 10 3
20-plex cell surface marker2 leukemic cell lines and
one patient sample
characteristic of undifferentiated
cells
THP-1 FAB M5
KG1a FAB M0
BCLQ Patient M5a
isotopically enriched tags
![Page 16: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/16.jpg)
Flow - ICP-TOF-MS
every cell ?
or pre-selected cells ?
![Page 17: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/17.jpg)
photo (“rear”) of Research Prototype CyTOF™ instrument
![Page 18: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/18.jpg)
Phase 0 CyTOF™ Prototype
![Page 19: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/19.jpg)
real time screen capture during data acquisitionKG-1a cellsfixed, Ir- intercalator CD7-139La CD13-144NdCD44-151EuCD45-159TbCD38-165Ho CD34-169Tm
m/z 139 144 151 159 165 169 191 193
Time Of Flight (mass)
scan
nu
mb
er (@12
s)
![Page 20: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/20.jpg)
Yb
175.
9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
fixed, Ir- intercalatorCD7-139La, CD13-144Nd,CD44-151Eu,CD45-159Tb,CD38-165Ho, CD34-169TmCD49d-176Yb
KG-1a cells multiplex detectionAugust 3, 2007research bread-board
![Page 21: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/21.jpg)
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Pr141
Eu
15
1
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Pr141
Eu
153
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Pr141
Tb
159
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Pr141
Ho
16
5
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Pr141
Tm
16
9
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu151
Eu
153
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu151
Tb
159
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu151
Ho
16
5
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu151
Tm
16
9
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu153
Tb
159
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu153
Ho
16
5
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Eu153
Tm
16
9
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Tb159
Ho
16
5
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Tb159
Tm
16
9
0
2
4
6
8
10
12
0 2 4 6 8 10 12
Ho165
Tm
16
9
151Eu
153Eu
159Tb
165Ho
169Tm
2-D plots for DTPA-metal labeled beads measured with ICP-MS-based cytometer for mixture of two bead types.
Ho, Tm beads(total 327 events)
Pr,Eu,Tb beads(total 461 events)
151Eu 153Eu 159Tb 165Ho141Pr
![Page 22: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/22.jpg)
m/z
m/z
O
AuGene A
Probe A
O
AuGene B
Probe B
Cell mRNA cDNA cDNA-Au(RT-PCR) (labeling)
(labeling)
mRNA-Au
0.8-3 mm
Polystyrene beads
Ru Rh
Dy
Ru
Ce
Gd
Au
Au
Next: massively multiplexed bead assay
30,000 distinguishable beads = 30-fold redundancy assay for
1000 genes@ 1kHz = 30 seconds
![Page 23: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/23.jpg)
http:/www/uhnres.utoronto.ca/studies/stemspec
MAXPAR™ reagents CyTOF™ analyzer
http://www.DVSsciences.com
![Page 24: Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky.](https://reader036.fdocuments.in/reader036/viewer/2022062518/56649d1f5503460f949f3e8c/html5/thumbnails/24.jpg)
Acknowledgements – funding
Genome Canada through the Ontario Genomics InstituteOntario Cancer Research NetworkMaterials and Manufacturing OntarioNational Institutes of HealthUniversity of TorontoMDS SciexDVS Sciences Inc.CytopeiaParker Life Sciences Leybold Vacuum GmBHETP division of SGECCR/MKS Process Products