Geng&started&on&the& CyTOF: learning&by&living&
Transcript of Geng&started&on&the& CyTOF: learning&by&living&
Ge#ng started on the CyTOF: learning by living
Alice Long and BRI HIP core team 2013
BRI Timeline
May Nov Feb Mar April Jan Dec
PracJce & Panel Development
PreparaJon
InstallaJon
Open for business Training
$
PreparaJon • Funding decisions
– External funding: Murdock foundaJon – Internal BRI funds to support HIP core CyTOF development
• Pro’s: – Not dependent on immediate grant funding – Jme to produce preliminary data for grants
• Con’s: – No solid commitment from staff to use the machine
• Gaining experience – Visited others faciliJes
• OHSU – Mandy Boyd • FHCRC – Andrew Berger • Lessons learned
– Instrument set-‐up: locaJon of CyTOF in lab, gas and vacuum flow – Re-‐iteraJon of instrument operaJon and troubleshooJng: common disposables
• EducaJon – PotenJal of technology
• BRI-‐wide and Faculty presentaJons – Comparison of flow vs CyTOF
Flow Cytometry vs. Mass Cytometry Technology Fluorescence flow
cytometry Mass Cytometry
Measurement Fluorescent probes Stable mass isotope probes
General CapabiliJes
20 parameters, 18 fluorescent (difficult)
37 ….and counJng (easy*)
-‐ Novel metals (i.e. barium, gold, iodine)
Fluorescent barcoding Metal tag barcoding
FSC and SSC -‐
Ca2+ flux -‐
Mitochondrial Assessment ?
Cell division (CFSE) -‐
Live cell sorJng -‐
Edited from Bendall S, et.al., Trends Immunol 2012
Flow Cytometry vs. Mass Cytometry Technology Fluorescence flow
cytometry Mass Cytometry
SensiJvity 0.1-‐10 1-‐2
QuanJtaJve Yes Yes
Throughput
Cells/s 25,000 1000 (500)
Cells/hr 25-‐60 million 1 million
Efficiency > 95% < 30%
Reagent Cost
Per probe per test $2.00-‐$8.00 $1.50-‐$3.00
Maintenance
Daily 15 minutes 60 minutes
Weekly 30 minutes 4 hours
InstallaJon and training
• Hands-‐on experience! • Use DVS support!
PracJce and Panel Development
• IniJal pracJce and troubleshooJng • Surface panel development – Ab selecJon – Ab conjugaJon – Sample tesJng
• Current development projects – Class II tetramer – Phospho and ICS panels built of current surface panel
– Mouse surface panel
IniJal pracJce and troubleshooJng • PracJce
– Start the CyTOF daily for a few weeks, someJmes running basic samples to become comfortable with maintenance and troubleshooJng
– Become familiar with sosware and manuals
Selected issues encountered solu9ons
Takes a few Jmes to start Make sure nebulizer is completely gassed-‐out
Pulse count, dual count, Tb TOF low (not pass QC)
SUPER clean copper contacts with EtOH
Sampler orifice damage? Installed replacement
No fcs file aser run Stopped early, but data retrievable
Mass calibraJon off Leslie from DVS fixed remotely
OxidaJon high OpJmize make-‐up and nebulizer gas flows
Other lessons learned
• Running samples – Importance of counJng cells and flow rate – OpJmizing protocols
• Reduce washes • Least amount of Jme in water • Stability of stained cells • Need for permebilizaJon and DNA labeling?
• Ask quesJons! – Thanks for help from DVS and Stanford team
DVS Raw Cytof Data
Really Raw Cytof Data
Development of a 28 Marker Mass Cytometry Panel
• Developed Metal Panel – Ordered 21 pre-‐conjugated human AnJbodies from DVS Sciences as closely matching Stanford panels as possible
– Conjugated 7 with MAXPAR® kits – Titrated all conjugates in the same tube on fresh and frozen PBMC
• Compared Panel to tradiJonal Flow Cytometry – 3 human subjects, Ficoll separated – Assayed 4 fluorescent panels – Assayed 1 metal panel – Collected on LSRII and CyTOF
PBMC Surface Phenotyping Panel
Ab label Ab label Ab label
CCR4 149Sm CD161 159Tb CD45RO 152Sm
CCR6 141Pr CD19 142Nd CD56 176Yb
CCR7 150Nd CD24 169Tm CD8 168Er
CD10 156Gd CD25 143Nd CRTh2 175Lu
CD11c 162Dy CD27 144Nd CXCR3 158Gd
CD123 151Eu CD3 170Er HLA DR 174Yb
CD127 171Yb CD38 167Er IgD 146Nd
CD14 160Gd CD4 145Nd IgM 172Yb
CD15 164Dy CD45 154Sm
CD16 165Ho CD45RA 153Eu
Typical JtraJon
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
0uL
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
0.03uL
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
0.06uL
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
0.125uL
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
0.25uL
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
0.5uL
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
1uL
0 101 102 103 104
(Yb172)Di: CD57
0
101
102
103
104
(Er1
70)D
i: C
D3
2uL
50 µL total volume
Use of isotype controls?
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
FSC-H
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105
<Pacific Blue-A>: CD3
0
50K
100K
150K
200K
250K
SSC
-A
0 30 60 90 120Cell_length
0
101
102
103
104
(Sm
154)
Di:
CD
45
0 101 102 103 104(Ir193)Di
0
101
102
103
104
(Ir191)Di
0 101 102 103 104
(Er170)Di: CD3
0
101
102
103
104
(Sm
154)
Di:
CD
45
Ga9ng for tradi9onal Flow Cytometry
Ga9ng for Mass Cytometry
0 102 103 104 105
<Qdot 605-A>: CD8
0
102
103
104
105
<V50
0-A
>: C
D4
0 103 104 105
<PerCP-Cy5-5-A>: CCR6
0
103
104
105
<PE-
Cy7
-A>:
CD
45R
O
0 103 104 105
<V500-A>: CD14
0
103
104
105
<Ale
xa F
luor
700
-A>:
HLA
-DR
0 101 102 103 104
(Nd145)Di: CD4
0
101
102
103
104
(Er1
68)D
i: C
D8
0 101 102 103 104
(Gd160)Di: CD14
0
101
102
103
104
(Yb1
74)D
i: H
LA-D
R0 101 102 103 104
(Pr141)Di: CCR6
0
101
102
103
104
(Sm
152)
Di:
CD
45R
O
Ga9ng for tradi9onal Flow Cytometry
Ga9ng for Mass Cytometry
Differences in MFI and double posiJve populaJons?
Comparison of Mass Cytometry to TradiJonal Flow Cytometry (FC)
Outliers CD38 CRTH2 IgD
Examples of SPADE trees from whole blood samples gate on live cells, cluster on CD19, CD4, CD8, CD3, CD123, CD56, CD11c
CD27 density CCR4 density
AnJgen Specific Cell Phenotyping (Kwok Lab)
• HA Clonal Cells/PBMC spiked in. • Cells surfaced labeled with AnJbody (2H11) and metal-‐tagged tetramer (StAv metal)
• Washed and labeled with 2H11-‐166Er • Fixed with 2% PFA • Permeablized with Saponin • Washed and labeled with Ir191,193 • Washed with MilliQ water • Resuspended in MQ and Eu151,153
Class II -‐ Flu Tetramer PosiJve
0 30 60 90 120Cell_length
0
101
102
103
104
(Sm
154)
Di:
CD
45
32.2
0 101 102 103 104
(Ir193)Di
0
101
102
103
104
(Ir19
1)D
i
95.7
0 101 102 103 104
(Nd145)Di: CD4
0
101
102
103
104
(Er1
70)D
i: C
D3
80.8
0 101 102 103 104
(Er166)Di: 2H11
0
101
102
103
104
(Nd1
45)D
i: C
D4
15.8
0 101 102 103 104
(Tb159)Di: Tmr1
0
101
102
103
104
(Nd1
45)D
i: C
D4
15.4
0 101 102 103 104
(Er166)Di: 2H11
0
101
102
103
104
(Tb1
59)D
i: Tm
r1
2.99 10.8
4.581.7
Tetram
er 1
Ir CD3
Cell Length
CollaboraJon with Kwok lab
CD4
CD4
CD45
Ir CD4
Tetramer 1 2H11 2H11
Current troubleshooJng with Class II Tmr
Issues Possible solu9ons
PermeablizaJon reduces Tmr binding, parJcularly for low
affinity interacJons
-‐ Eliminate permeablizaJon and idenJfy cells by surface markers
Low frequency of Class II Tmr+ cells in PBMC
-‐ PosiJvely select Class II Tmr+ cells using anJ-‐PE Miltenyi columns
-‐ Amplify signal using Strepavidin conjugated heavy metal or anJ-‐bioJn-‐metal Ab
-‐ Use surrogate marker of Tmr for iniJal tests of low frequency events
c-‐myc Class II Tmr producJon -‐ Trial and error
Current troubleshooJng with ICS and phoshpo stains
Issues Possible solu9ons
Pairing nuclear stains with ICS
-‐ TesJng BD transcripJon factor buffer by flow for mulJple targets (flow vs CyTOF for nuclear stains)
-‐ TesJng different FOXP3 anJbodies
SelecJon of best heavy metal for bar-‐coding
-‐ Trial and error
Best level of sJmulaJon to detect subtle changes in autoimmune diseases
-‐ Determining ½ maximal and maximal sJm for phospho and ICS
-‐ SelecJon of posiJve control samples (PMA/I, cytokine capture techniques, lines and clones)
Open for Business
• BRI Resources – HIP core services
• Maintenance of CyTOF • Scheduling CyTOF use
– HIP core resources • Advice on experimental/panel design • Advice on protocols
– Provide protocols and training • Metal conjugated Ab • AcquisiJon of data • FloJo QC and SPADE analysis of data
Cost structure Reagents acquisi9on FloJo
analysis SPADE analysis
BRI cost/sample
Tier 1
HIP core HIP core HIP core $240
Tier 1 (SPADE)
HIP core HIP core HIP core HIP core $300
Tier 2 (trained
personnel)
BRI and/or HIP core ($300/vial) or $8/test
HIP core $200/hr
BRI or HIP core
$50/hr for
analysis-‐training
Tier 3 (specially trained
personnel)
BRI and /or HIP core
BRI $100/hr
BRI
SPADE is $1,200/year/user
CyTOF users to-‐date
• HIP core – Ian Frank – Jerill Thorpe – Katharine Schwedhelm – Alice Long
• Kwok lab for Tmr stains • Jane Buckner and Alice Long 1st customers – Phospho and ICS stains on clinical samples
BRI Inves9gators Jerry Nepom
Bill Kwok Erik Wambre Jane Buckner
Funding Support NIAID JDRF Murdock FoundaJon
Human Immunophenotyping Core (HIPc) at BRI S. Alice Long
Katharine Schwedhelm Jerill Thorpe
Ian Frank
NIAID