Transcript 2100 Bioanalyzer Applications Master Jun_07

Overview on
Agilent Lab on a Chip
Applications
Page 1
2100 Bioanalyzer Solutions
March 2007
Group/Presentation Title
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Month ##, 200X
The Lab-on-a-Chip Approach
Active Control of Fluids without Moving Parts
Sample volumes 1 -4 µl
10 -12 samples depending on Assay
Separation, staining, detection of samples
Results in 5-30 minutes available
No extra waste removal needed
Disposable Chip, no crosscontamination
Page 2
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Lab-on-a-Chip or Gel - A Faster Answer !
Page 3
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Agilent 2100 Kits and consumables
Various Kits and consumables are available
to support the predefined methods o the
2100 Bioanalyzer
Agilent RNA 6000 Nano Kit
Agilent RNA 6000 Nano Reagents
Agilent RNA Nano Ladder
Agilent RNA 6000 Pico Kit
Agilent RNA 6000 Pico Reagents
Agilent RNA Pico Ladder
Agilent DNA 1000 Kit
Agilent DNA 1000 Reagents
Agilent DNA 7500 Kit
Agilent DNA 7500 Reagents
Agilent DNA 12000 Kit
Agilent DNA 12000 Reagents
Agilent Protein 230 Kit
Agilent Protein 230 Reagents
Agilent Protein 80 Kit
Agilent Protein 80 Reagents
Agilent Cell Kit
Agilent Cell Checkout Kit
Page 4
5067-1511
5067-1512
5067-1529
5067-1513
5067-1514
5067-1535
5067-1504
5067-1505
5067-1506
5067-1507
5067-1508
5067-1509
5067-1517
5067-1518
5067-1515
5067-1516
5067-1519
5067-1520
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RNA Applications
RNA QA/QC
for
Microarrays
Gene
Expression
RNA QA/QC
for qPCR
RNA QA/QC
for mPCR
Page 5
Genomic
DNA
Contaminatio
n
smallRNA
QA/QC
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Specifications RNA assays (Series2 assays)
RNA 6000 Nano LabChip kit
Analytical
Specification
Qualitative Range
Quantitative Range
total RNA
mRNA
5-500ng/ul
25-500ng/ul
25-250ng/ul
25-250ng/ul
Reproducibility of
Quantitation
10% CV
10% CV
Maximum Sample
Buffer Strength 10 mM Tris-EDTA 10 mM Tris-EDTA
Physical
Specification
Analysis Time
Number of
Samples
Sample Volume
Assay Kit Stability
Page 6
30 minutes
30 minutes
12 samples/chip
1ul
4 months at 4 C
12 samples/chip
1ul
4 months at 4 C
RNA 6000 Pico LabChip kit
Analytical
Specification
Qualitative Range
Maximum Sample
Buffer Strength
total RNA
mRNA
50-5000 pg/µl
250-5000 pg/µl
50 mM Tris or NaCl 50 mM Tris or NaCl
Physical
Specification
Analysis Time
Number of
Samples
Sample Volume
Assay Kit Stability
30 minutes
30 minutes
11 samples/chip
1 µl
4 months at 4 C
11 samples/chip
1 µl
4 months at 4 C
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Features of the RNA 6000 Assays
total RNA
determine integrity and quality of total RNA
determination of RNA concentration
identify ribosomal peaks
calculate the ratio of ribosomal peaks (18S/28S or
16S/23S)
RNA integrity number (RIN)
mRNA
determine integrity and quality of mRNA samples
Determination of mRNA concentration
calculate % ribosomal RNA in mRNA samples
Page 7
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RNA 6000 Nano LabChip kit
Analysis of Total RNA Integrity
11
18S
10
28S
Typical first QC step after
RNA sample prep prior to
microarrays or real-time
PCR
9
Fluorescence
8
7
6
marker
5
4
High quality total RNA
3
2
0
19
24
29
34
39
28S
18S
1
44
49
54
59
64
69
Time (seconds)
11
10
9
8
Fluorescence
7
6
5
Partially degraded total RNA
marker
4
3
2
0
19
24
29
34
39
28S
18S
1
44
49
Time (seconds)
2100 bioanalyzer:
electropherogram
Page 8
54
59
64
69
2100 bioanalyzer: single lane gel-like
image
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RNA QC in Routine Gene Expression Workflow
Cells / Culture
RNA isolation
Start again with
sample isolation
Total RNA
RNA QC via Agilent 2100 bioanalyzer
RIN
RIN above threshold
Continue with downstream Experiment (Microarray, real-time PCR, etc.)
Page 10
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RIN Application – Directly Compare Samples
same sample in different dilutions
12.5
25 ng/µl: RIN 8
Fluorescence
10.0
7.5
5.0
0.0
28S
18S
2.5
20
Fluorescence
When testing an identical
RNA sample in various
dilutions, identical RINs
are obtained – within
narrow limits
108 samples 3 dilutions
CV RIN: 3 %
CV ribosomal ratio: 22 %
100 ng/µl: RIN 8
15
10
0
28S
18S
5
80
500 ng/µl: RIN 8
Fluorescence
70
60
50
40
30
20
0
19
24
29
34
39
28S
18S
10
44
49
54
59
64
69
Time (seconds)
Page 11
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RIN Application – Assessment of RNA Integrity
45
Fluorescence
40
35
30
Intact RNA: RIN 10
25
20
15
18S
5
0
28S
10
9
Fluorescence
8
7
6
Partially degraded RNA: RIN 5
5
4
3
2
0
28S
18S
1
3.5
Fluorescence
3.0
2.5
2.0
Strongly Degraded RNA: RIN 3
1.5
1.0
0.5
0.0
19
24
29
34
39
44
49
54
59
64
69
Time (seconds)
Page 12
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Mouse kidney II mRNA
Mouse kidney I mRNA
Mouse liver mRNA
Mouse testis mRNA
Bovine kidney mRNA
Rat brain mRNA
RNA 6000 ladder
Ribosomal RNA contamination in mRNA samples
12.5
rRNA contamination: 19.5 %
10.0
Fluorescence
7.5
5.0
18S
0.0
16
21
26
31
28S
2.5
36
41
46
51
56
61
66
71
76
81
Time (seconds)
5.5
5.0
4.5
rRNA contamination: 1.7 %
4.0
3.5
Fluorescence
3.0
2.5
2.0
1.5
1.0
0.5
28S
18S
0.0
-0.5
21
26
31
36
41
46
51
56
61
66
71
76
81
Time (seconds)
Page 13
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Laser Microdissection – PALM MicroBeam System
A
200 pg
total
RNA
Laser
microdissection
Catapulted
area in the
collection
device
28S
24 29 34 39 44 49 54 59 64 69
C
Laser Microdissection and Pressure
Catapulting (LMPC)
Page 14
18S
B
Laser pressure
catapulting:
Section after
catapulting of
selected area
RNA extraction
RNA sample QC using the Agilent 2100
bioanalyzer and the RNA 6000 Pico LabChip kit
Data kindly provided by P.A.L.M.
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MicrolaserMonth
Technologies
cRNA Hybridization - Workflow
mRNA QC
Total RNA QC
Cy3/ Cy5 Labeling
Array experiment
Data evaluation
cRNA fragmentation
Page 15
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Series II RNA Assays
A new RNA Ladder is now included in the kit.
Improved quantitation accuracy with salt containing samples. No late
migration of 28S peaks
Pico: higher sensitivity, better reproducibility and accuracy in quantitation
new
old
- Higher reagent volumes at no charge
Page 16
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RNA Con’t
Increased sensitivity, Pico Assay
50 pg/µL total RNA in water (Series II
RNA 6000 Pico assay)
Page 17
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Analysis of Small RNA (using RNA 6000 Assay)
Small RNA
fraction: < 200 nts
e.g. miRNA,
siRNA, snRNA,
tRNA, 5S RNA
Bioanalyzer
allows
discrimination of
different profiles
Page 18
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Small RNA Assay specifications
Analytical Range
6 -150 nt (to avoid overlap)
Sensitivity
50 pg/µl
(diluted Ladder - 40 nt fragment; S/N > 3:1)
Quantitative range
50 pg/µl – 2000 pg/µl
(purified miRNA in water after extraction ~<200nt)
Quantitation Reproducibility
25 % CV
(defined on Ladder)
Max amount total RNA
100 ng/µl total RNA
Carryover
Below detection limit
Page 19
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18s rRNA
tRNA
RNA 6000 Assay
Two complimentary
assays, one to evaluate
total RNA integrity and
the second to
characterize the small
RNA fraction
Small RNA Assay
Small
rRNAs
tRNA
miRNA
Page 20
28s rRNA
Lower Marker
[nt]
Lower Marker
50bp
New Small RNA Assay
Fragments larger than
200nt (i.e. 18S/28S) are
not injected and do not
interfere with the
assay
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28s
Small RNA Region
Lower Marker
18s
New Small RNA Assay versus existing RNA Assay
RIN: 8.1
RNA 6000Nano
Size range: 25-6000nt
Results: Integrity, Total RNA amount, gDNA
contamination
RNA 6000Nano kit
Small RNA Kit
Page 21
5.8s
tRNA
5s
miRNA Region
Lower Marker
Small RNA Region
NEW! Small RNA
Size range: 6-150nt
Results: miRNA amount, Ratio and amount of
other Small RNA
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Protein Applications
Protein
QA/QC
Antibody
QA/QC
Compliant
Protein &
Antibody
QA/QC
Protein
Expression
Food
Analysis
Protein
Purification
Page 22
Group/Presentation Title
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Month ##, 200X
Protein LabChip Kits – Specifications (Series II)
• Automated analysis of 10 protein samples in less than 30
minutes
• Two assays for complementary size ranges
– Protein 80:
5 to 80 kDa
– Protein 230: 14 to 230 kDa
• Sizing resolution of 10% across the size range
• Large linear dynamic range
(e.g. from 15 - 2000 ng/l CA-II in PBS)
• Sensitivity equivalent to non-colloidal Coomassie (R-250) stain
• Relative and absolute quantitation
• Compatible to a variety of sample buffer components
Page 23
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Protein Series II Assay - Specs
Parameter
Analysis time [min]
No of samples
Sample vol [µl]
Sizing range [kDa]
Typical sizing resolution
Typical sizing accuracy
Sizing reproducibility
Sensitivity CAII
Sensitivity BSA
Quantitative range [ng/µl] CAII
Qualitative range [ng/µl] CAII
Quantitation reproducibility
Page 24
Protein 230
Protein 80
25
10
4
30
10
4
14 - 230
10%
10% CV
3% CV
6ng/µl
15ng/µl
15 - 2000
6 - 5000
20% CV
5 - 80
10%
10% CV
3% CV
6ng/µl
15ng/µl
60 - 2000
6 - 4000
20% CV
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Staining, Destaining and Detection
Staining,
Destaining and Detection
detection
X
If no dilution was done the
micelles would result in
high background and low
sensitivity
SDS + dye
destai
n
detectio
n
protein
micelles
Page 25
low background good
signal to noise ratio SDS
conc. below CMC
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Staining, Destaining and Detection
Staining,
Destaining and Detection
SDS + dye
protein
micelles
destai
n
detectio
n
low background good
signal to noise ratio SDS
conc. below CMC
Page 26
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Protein LabChip Applications
Cell lysates
• identification of over-expressed proteins
• comparison of different expression patterns
Column fractions
• monitoring of protein isolation and purification process
• check fractions for impurities
Purified proteins
• monitoring of impurities in protein preps
• integrity check for monoclonal or polyclonal antibodies
(antibody QC)
Page 27
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Clone Selection based on Protein Expression
Example measured with
Protein 50 kit
colony 1
60
53.0
50
6.0
10
0
20
Page 28
25
30
35
40
45
Time (seconds)
8*
20
6
7
14.4
4
5
protein of interest
30
2
3
21.5
40
1*
32.5
29.0
Fluorescence
kDa
colony 2
50
55
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Monitoring of Protein Purification Process
Example measured with
Protein 200 plus kit
GFP Fusion Protein
Analysis
cell lysate
flow through
(fraction 66)
wash
(fraction 69)
elution
(fraction 77)
wash
(fraction 78)
15
2100 bioanalyzer: gel-like image
Page 29
20
25
30
35
Time (seconds)
2100 bioanalyzer: electropherogram
40
45
Courtesy of P. Sebastian and S.R. Schm
GPC-Biotech AG, Martinsried, German
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Protein P230 Series II Assay
Assay reworked for optimized stability
– Sizing range from 14-230 kDa
– UM at 240 kDa
– New lower marker and optimized LM detection
(less overlap with system peak)
– New ladder
– Baseline stability and better sensitivity
High purity UM and ladder improves usability
in QC (optimized for low level impurity
quantification -> compliance).
Page 30
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Quality Control of Antibodies
Antibody analysis under reducing and non-reducing
conditions
Ab reduced
heavy
chain
intact
antibody
light chain
Determine the half antibody content in IgG
intact
preparations
antibody
Ab nonreduced
light + heavy
chain
16% half
antibody
90 kDa
Page 31
160 kDa
Absolute Quantitation of IgG samples
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Protein 80 Series II assay
• sizing range 5-80 kDa (specific fit to antibody QC)
• new ladder & new UM
• improvements in sensitivity (sizing/quantitation)
• improvements in sensitivity specifications as P230
ladder run
Page 32
PBS blank run
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2100 Bioanalyzer Compliance
2100 expert software
- One version for all assays
- Declaration of system validation
2100 expert security pack
- 21 CFR part 11compliance
- Electronic records
- Electronic signatures
- Audit trails
2100 bioanalyzer
- IQ and OQ/PV services
- Declaration of conformity
Chips and reagents
- Declaration of conformity
Page 33
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Month ##, 200X
DNA Applications
Forensic
Testing
Food
Analysis
mtDNA
Screening
qPCR
validation,
impurity
check
mPCR
validation,
impurity
check
Restriction
Digest
Analysis
Gene
Expression
Clinical
Research
Page 34
Oncology
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Application Areas for the DNA Assays
PCR product purity
Multiplex PCR Applications
Gene expression analysis via RT-PCR (target validation)
GMO testing
Pathogen detection (homeland defense, hospitals,
environmental)
Genotyping applications
• Duplications/ deletions
• Allele frequency
• Bacterial sub-typing
• Forensics
Cancer diagnostics
Page 35
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Data Format - Gel-Like Image c/w Agarose Gel
marker
900, 1000 bp
900, 1000 bp
473, 500 bp
300, 315 bp
473, 500 bp
300, 315 bp
100, 105 bp
100, 105 bp
25 bp
marker
2100 bioanalyzer data
Gel-like image
Page 36
25 bp
2 % agarose gel stained
with Ethidiumbromide
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Determination of PCR Product Impurity
Agilent 2100 bioanalyzer
1
3000 bp
2
3
4
Agarose gel
1
5
2000
1200
800
400
300 bp
2
3
4
5
2000
1200
800
400
200
100
200
100
Page 37
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Determination of PCR Product Impurity
300 bp PCR
300 bp PCR 1:4 dil.
300 bp
500
Quantitative data from Agilent 2100 bioanaly
Impurity level : < 2%
400
Sample
Fluorescence
300
300 bp PCR
300 bp PCR 1:4
200
25
30
2
3
1*
0
35
40
45
main peak
41.4 ng/ul
9.6 ng/ul
40.7 ng/
9.6 ng/u
4*
100
c (DNA)
50
55
60
65
70
75
80
85
90
Time (seconds)
3000 bp PCR
500
3000 bp PCR 1:4 dil.
Quantitative data from Agilent 2100 bioanaly
3000 bp
Impurity level : > 50%
400
Sample
Fluorescence
300
3000 bp PCR
3000 bp PCR 1:4
200
25
30
35
40
45
50
55
60
65
70
75
80
main peak
61.9 ng/ul
14.8 ng/ul
40.7 ng/
9.8 ng/
7*
4
5
6
2
1*
0
3
100
c (DNA)
85
90
Time (seconds)
Page 38
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GMO Detection: Determination of GM Soya
Percentage
EPSPS gene target: specific
for Roundup Ready GM soya
(Monsanto)
Soya lectin
gene target
Data kindly provided by CCFR
Page 39
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Month ##, 200X
Effect of Multiplex PCR buffer on a 19-plex PCR
Data kindly provided by QIAGEN GmbH, Germany
Page 40
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Month ##, 200X
Tumor Diagnostics
1. Spiking experiment with given amount of
cancer cells
2. Enrichment with AdneGen Cancer Select
kit (antibody based immunomagnetic
enrichment.)
3. Multiplex Amplification with AdnaGen
CancerDetect kit
4. Detection with Agilent 2100 Bioanalyzer
Data kindly
and DNA 500 LabChip
kit provided by Adnagen
Page 41
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Month ##, 200X
Detection of Single Base Mutations (1)
in Exons 7 and 8 of the Human p53 Gene by RFLP Mapping using the DN
exon 7
exon 8
Amplify exons 7 and 8 (resulting
products:
618 bp fragment and 200 bp
fragment)
Digest with Hpa II
In each example one of the restriction
83 bp, 91 bp, 91 bp, 109 bpsites can be deleted by a point mutatio
168 bp, 276 bp
Analyze using Agilent 2100 bioanalyz
and 4-20 % acrylamide gel
Page 42
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Detection of Single Base Mutations (2)
p53Exon 8 wt/HpaII
p53Exon 8 clone 106/HpaII
Upper
Marker
Fluorescence
25
Lower
Marker
20
wt 106
111 bp
15
208 bp
90 bp
10
109 bp
200 bp
5
1*
2
3
4*
91 bp
0
25
30
35
40
45
50
55
60
65
Time (seconds)
30
251 bp
Lower
Marker 84/85 bp
Upper
Marker
267/268 bp
276 bp
15
276 bp
5
91+83 bp
30
35
4
40
251 bp
91 bp
5*
168 bp
3
10
0
59
166 bp
20
1*
2
Fluorescence
25
wt
p53Exon 7 clone 59/HpaII
p53Exon 7 wt/HpaII
45
50
55
60
65
70
75
Time (seconds)
Page 43
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Month ##, 200X
Label free Analysis of Microsatellite Instabilities
Clinical Diagnostics and Molecular Diagnostics of Cancer
Microsatellite instabilities
present in 10-15% of colon
and gastric carcinomas
Study: 40 cases of colon
carcinoma
5 microsatellite loci
investigated
Results compared with
traditional PAGE:
95% concordance
rate
Page 44
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Month ##, 200X
Cell Applications
Protein
Expression
Monitoring
Intracellular
Apoptosis
Detection
Gene
Silencing
Protein
Expression
Monitoring
Cell Surface
Antibody
Staining
Page 45
Transfection
Efficiency
Monitoring
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Cell Assay
Apoptosis
Transfection Efficiency Monitoring
•
•
Detection of GFP-transfected cells
Antibody staining: Detection of transfected cells expressing the
encoded protein
Protein Expression Monitoring
•
Extracellular and Intracellular Antibody staining for detection of protein
expressed on the cell surface, in the cytoplasm, or in the nucleus
Gene silencing
Page 46
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Principle of Pressure-Driven Flow
For cell assays (analysis of cell fluorescence parameters)
On-chip simple flow cytometric studies
Pressure driven
flow is used to
move cells in a
controlled
manner through
the microchannels
Cells are
hydrodynamica
lly focused to a
portion of the
channel by a
side stream of
buffer
Cells pass the
fluo-rescence
detector in single
file and each event
is monitored in a
histogram or dot
plot
The microchannels of
the glass chip
are filled with
cell buffer
Page 47
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The Bioanalyzer Lab-on-a-Chip
Approach
Separation on disposable, µ-fabricated glass
chips
made of two glass layers:
• one with micro-channels (x10µm, etched),
• one with through-holes
• glued into a plastic caddy which accommodates
wells for gel, sample, standard (ladder), buffer and
other reagents
• for handling nl-amounts of liquids
• one separation channel for ladder and sample
• microfluidic sample movement with fluorescence
detection
•
Setup
• micro-channels are filled with gel or buffer
• sample, ladder and reagents are filled into the
respective wells
•chip preparation in less than 5 minutes
Benefits
• convenient handling
• minimized risk of cross-contamination
• versatile design for multiple experiments on one
platform
Page 48
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Flow Cytometry on a Chip - Hydrodynamic
Focusing
All six cell samples are
hydrodynamicly focused to one side
of the micro channel
At each of the six pinch points the cell
stream is joined by a buffer stream
from one of the two buffer wells
The two liquids do not mix
immediately
The cells then move towards the
detector in single file
Page 49
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Flow Cytometry on a Chip
- Two Color Detection- Three Types of Events
Red cells
Blue/red cells
Blue cells
Dot plot view for easy
data evaluation
Page 50
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Some Target applications
Apoptosis:
Annexin V
Caspase-3
Detection of phosphatidylserine on the cell surface
Detection of activated caspase-3 in the cytoplasm
Transfection Efficiency Monitoring:
GFP:
Detection of GFP-transfected cells
Antibody staining: Detection of transfected cells expressing the
encoded
protein
Protein Expression Monitoring:
Extracellular and Intracellular Antibody staining for detection of
protein expressed on the cell surface, in the cytoplasm, or in the
nucleus
Gene silencing:
Optimization of siRNA transfection procedure
Verify silencing by cellular protein expression measurement
Correlation of siRNA uptake and gene knockdown
Page 51
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Cell assays: sample preparation
Typical workflow:
Customer
Agilent
Add staining reagent
and incubate
adherent
(trypsinize) & harvest
suspensionby centrifugation,
wash
wash twice
Resuspend cells in
cell buffer (LabChip Kit)
Data analysis
to result
Page 52
Load on chip
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Flow Cytometry on a Chip - Optics & Detection
2100 Bioanalyzer
Red detection channel:
• 620-645 nm excitation with Laser (Maximum 630 nm)
• 674-696 nm detection range (Maximum 680 nm)
Blue detection channel:
• 458-482 nm excitation with LED (Maximum 470 nm)
• 510-540 nm detection range (Maximum 525 nm)
Standard Flow Cytometers have 3-4 fluorescence detection channels:
FL1: excitation 488 nm, detection 530 nm
FL2: excitation 488 nm, detection 585 nm
FL3: excitation 488 nm, detection 661 nm
FL4: excitation 635 nm, detection 670 nm
Page 53
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Cell Assays - Applications: Apoptosis
Annexin Binding
Healthy cell
Dead cell
Live dye: Calcein
biotin-Annexin+ Cy5streptavidin
“Live” apoptotic cell
Phosphatidyl-serine from inner leaflet flips to outer membrane
during apoptosis and can be labeled by Annexin V
Page 54
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Annexin V Assay (24h Induction)
Three Bioanalyzer instruments vs a flow cytometer reference instrum
5 chips, each loaded with control in well 1 and 24h sample in wells 2-6
% Apoptotic
80%
Chip 1
60%
Chip 2
Chip 3
Chip 4
Chip 5
2100-1
2100-2
2100-3
40%
Flow
cytometer
20%
0%
0
10
20
30
Sample Number
Page 55
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Applications: Protein Expression Analysis
GFP Transfection Efficiency Control
CHO-K1 cells were transfected with
EGFP DNA and Lipofectamine.
Mock transfected cells
0.1 %
Control
GFP transfected cells
56.6 %
EGFP
transfected
Disclaimer. Neither Agilent nor Caliper makes any representation that use of EGFP and
practice of the methods described in this publication is licensed or
otherwise authorized under any third party patents, including patents owned
or exclusively licensed by Aurora Biosciences or Amersham Biosciences.
Page 56
Group/Presentation Title
Agilent Restricted
Month ##, 200X
GFP Transfection Efficiency
Transfection Efficiency
70%
60%
50%
Flow cyt.
40%
2100-1
2100-2
2100-3
30%
20%
10%
0%
1
2
3
4
5
Chip Number
Page 57
ctrl
mean
SD
2100-1
0.46
0.08
GFP
mean
SD
%CV
60.19
2.13
3.54
2100-2
0.31
0.29
2100-3
0.47
0.43
59.15
2.48
4.19
59.26
2.10
3.54
All
0.40
0.29
Flow cyt.
0.16
0.12
59.53
2.26
3.80
60.90
1.22
2.01
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Flow Cytometry Assays Applications - Cell
surface Antibody staining
Cell expressing
protein of interest
Cell not expressing
protein of interest
Target protein
Live dye: Calcein
Cy5 or APC-labeled Antibody
Page 58
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Extracellular Antibody Staining
Averaged data per instrument
80.00
% gated 2100-1
70.00
% gated 2100-2
60.00
% gated 2100-3
% gated
50.00
%gated 2100-4
40.00
Flow cytometer
30.00
20.00
10.00
0.00
1
2
3
4
sample #
Mean % CD3+ cells
2100-1
60.9
34.4
17.3
8.9
5.1
0.8
Page 59
2100-2
67.8
36.7
17.6
9.4
4.4
0.6
2100-3
66.6
36.7
18.7
9.9
5.3
0.3
2100-4
65.0
34.3
17.2
8.3
4.9
0.3
5
Flow cyt.
60.9
29.8
13.8
6.5
3.2
0.0
6
Jurkat cells were
stained with calcein
alone or with calcein
and APC-labeled
anti-CD3 antibody.
Mixtures of both
populations were
prepared at various
ratios.
Samples were
analyzed with four
2100 instruments on
5 chips and
compared to a flow
cytometer reference
instrument
Group/Presentation Title
Agilent Restricted
Month ##, 200X
GFP On-Chip Staining - Workflow
35 min
Conventional
10
15min
min
10 min
10 min
Resuspend cells in CB
CBNF
Spin, aspirate,
resuspend,
spin, aspirate
On-chip
15 min
15 min
cells + CBNF
Page 60
Group/Presentation Title
Agilent Restricted
Month ##, 200X
GFP On-Chip Staining - Histogram Quality
2100 bioanalyzer
Flow
Cytometer
Page 61
Group/Presentation Title
Agilent Restricted
Month ##, 200X