Group/Presentation Title Agilent Restricted Month ##, 200X Page 1 Overview on Agilent Lab on a Chip...
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Transcript of Group/Presentation Title Agilent Restricted Month ##, 200X Page 1 Overview on Agilent Lab on a Chip...
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 1
Overview on Agilent Lab on a Chip
Applications
2100 Bioanalyzer SolutionsMarch 2007
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 2
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 3
Lab-on-a-Chip or Gel - A Faster Answer !
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 4
Agilent 2100 Kits and consumables
Various Kits and consumables are availableto support the predefined methods o the2100 BioanalyzerAgilent RNA 6000 Nano Kit 5067-1511Agilent RNA 6000 Nano Reagents 5067-1512 Agilent RNA Nano Ladder 5067-1529Agilent RNA 6000 Pico Kit 5067-1513 Agilent RNA 6000 Pico Reagents 5067-1514Agilent RNA Pico Ladder 5067-1535Agilent DNA 1000 Kit 5067-1504 Agilent DNA 1000 Reagents 5067-1505 Agilent DNA 7500 Kit 5067-1506Agilent DNA 7500 Reagents 5067-1507Agilent DNA 12000 Kit 5067-1508Agilent DNA 12000 Reagents 5067-1509Agilent Protein 230 Kit 5067-1517Agilent Protein 230 Reagents 5067-1518Agilent Protein 80 Kit 5067-1515Agilent Protein 80 Reagents 5067-1516Agilent Cell Kit 5067-1519Agilent Cell Checkout Kit 5067-1520
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 5
RNA Applications
RNA QA/QC for
Microarrays
Gene Expression
Genomic DNA
ContaminationRNA QA/QC
for qPCR
RNA QA/QC for mPCR
smallRNA QA/QC
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 6
Analytical Specification total RNA mRNA
Qualitative Range 5-500ng/ul 25-250ng/ulQuantitative Range 25-500ng/ul 25-250ng/ul
Reproducibility of Quantitation 10% CV 10% CVMaximum Sample
Buffer Strength 10 mM Tris-EDTA 10 mM Tris-EDTA
Physical Specification
Analysis Time 30 minutes 30 minutesNumber of Samples 12 samples/chip 12 samples/chipSample Volume 1ul 1ulAssay Kit Stability 4 months at 4 C 4 months at 4 C
Specifications RNA assays (Series2 assays)
Analytical Specification total RNA mRNA
Qualitative Range 50-5000 pg/µl 250-5000 pg/µlMaximum Sample
Buffer Strength 50 mM Tris or NaCl 50 mM Tris or NaCl
Physical Specification
Analysis Time 30 minutes 30 minutesNumber of Samples 11 samples/chip 11 samples/chipSample Volume 1 µl 1 µlAssay Kit Stability 4 months at 4 C 4 months at 4 C
RNA 6000 Nano LabChip kit RNA 6000 Pico LabChip kit
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 7
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
Features of the RNA 6000 Assays
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 8
RNA 6000 Nano LabChip kit Analysis of Total RNA Integrity
High quality total RNA
Partially degraded total RNA
18S
28S
Flu
ores
cenc
e
Time (seconds)
0
1
2
3
4
5
6
7
8
9
10
11
19 24 29 34 39 44 49 54 59 64 69
marker18
S
28S
Flu
ores
cenc
e
Time (seconds)
0
1
2
3
4
5
6
7
8
9
10
11
19 24 29 34 39 44 49 54 59 64 69
marker
28S18S Typical first QC step after RNA sample prep prior to microarrays or real-time PCR
2100 bioanalyzer: electropherogram
2100 bioanalyzer: single lane gel-like image
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 10
RNA QC in Routine Gene Expression Workflow
Start again with sample isolation
Cells / Culture
RNA isolation
Total RNA
RIN
RNA QC via Agilent 2100 bioanalyzer
RIN above threshold
Continue with downstream Experiment (Microarray, real-time PCR, etc.)
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 11
108 samples 3 dilutionsCV RIN: 3 %CV ribosomal ratio: 22 %
108 samples 3 dilutionsCV RIN: 3 %CV ribosomal ratio: 22 %
RIN Application – Directly Compare Samplessame sample in different dilutions
18S
28S
Flu
ores
cenc
e
0.0
2.5
5.0
7.5
10.0
12.5
18S
28S
Flu
ores
cenc
e
Time (seconds)
0
10
20
30
40
50
60
70
80
19 24 29 34 39 44 49 54 59 64 69
18S
28S
Flu
ores
cenc
e
0
5
10
15
20
25 ng/µl: RIN 8
100 ng/µl: RIN 8
500 ng/µl: RIN 8
When testing an identical RNA sample in various dilutions, identical RINs are obtained – within narrow limits
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 12
RIN Application – Assessment of RNA Integrity
Intact RNA: RIN 10
Partially degraded RNA: RIN 5
Strongly Degraded RNA: RIN 3
18S
28S
Flu
ores
cenc
e
0
1
2
3
4
5
6
7
8
9
18S
28S
Flu
ores
cenc
e
0
5
10
15
20
25
30
35
40
45
Flu
ores
cenc
e
Time (seconds)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
19 24 29 34 39 44 49 54 59 64 69
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 13
18S
28S
Flu
ores
cenc
eTime (seconds)
0.0
2.5
5.0
7.5
10.0
12.5
16 21 26 31 36 41 46 51 56 61 66 71 76 81
18S
28S
Flu
ores
cenc
e
Time (seconds)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
21 26 31 36 41 46 51 56 61 66 71 76 81
rRNA contamination: 19.5 %
rRNA contamination: 1.7 %
Mou
se k
idne
y I
mR
NA
Mou
se k
idne
y II
mR
NA
Rat
bra
in m
RN
A
Bov
ine
kidn
ey m
RN
A
Mou
se t
esti
s m
RN
A
Mou
se li
ver
mR
NA
RN
A 6
000
ladd
er
Ribosomal RNA contamination in mRNA samples
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 14
Laser Microdissection and Pressure Catapulting (LMPC)
RNA extraction
A
B
C
RNA sample QC using the Agilent 2100 bioanalyzer and the RNA 6000 Pico LabChip kit
Laser Microdissection – PALM MicroBeam System
Data kindly provided by P.A.L.M. Microlaser Technologies
Laser microdissection
Laser pressure catapulting: Section after catapulting of selected area
Catapulted area in the collection device
18S
28S
24 29 34 39 44 49 54 59 64 69
200 pg total RNA
200 pg total RNA
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 15
cRNA fragmentation
Cy3/ Cy5 Labeling
Total RNA QC
mRNA QC
Array experiment
cRNA Hybridization - Workflow
Data evaluation
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 16
Series II RNA AssaysA 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
- Higher reagent volumes at no charge
new old
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 17
RNA Con’tIncreased sensitivity, Pico Assay
50 pg/µL total RNA in water (Series II RNA 6000 Pico assay)
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 18
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 19
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 20
miR
NA
tRN
A
Small rRNAsL
ower
Mar
ker
[nt]
New Small RNA Assay
Fragments larger than 200nt (i.e. 18S/28S) are not injected and do not interfere with the assay
Lo
we
r M
ark
er
50
bp
tRNA
18s rRNA 28s rRNA
RNA 6000 Assay
Small RNA Assay
Two complimentary assays, one to evaluate total RNA integrity and the second to characterize the small RNA fraction
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 21
Sm
all
RN
A R
eg
ion
New Small RNA Assay versus existing RNA Assay
miR
NA
Re
gio
n
Lo
wer
Mar
ker
Small RNA Region
tRN
A
18s
28s
RIN: 8.1
Lo
wer
Mar
ker
RNA 6000Nano kit
Small RNA Kit
RNA 6000Nano
Size range: 25-6000nt
Results: Integrity, Total RNA amount, gDNA contamination
NEW! Small RNASize range: 6-150nt
Results: miRNA amount, Ratio and amount of other Small RNA
5s
5.8s
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 22
Protein Applications
AntibodyQA/QC
Protein Expression
Protein Purification
Food Analysis
ProteinQA/QC
Compliant Protein & AntibodyQA/QC
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 23
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 24
Protein Series II Assay - Specs
Parameter Protein 230 Protein 80
Analysis time [min] 25 30No of samples 10 10Sample vol [µl] 4 4
Sizing range [kDa] 14 - 230 5 - 80Typical sizing resolution 10% 10%Typical sizing accuracy 10% CV 10% CVSizing reproducibility 3% CV 3% CVSensitivity CAII 6ng/µl 6ng/µlSensitivity BSA 15ng/µl 15ng/µlQuantitative range [ng/µl] CAII 15 - 2000 60 - 2000Qualitative range [ng/µl] CAII 6 - 5000 6 - 4000Quantitation reproducibility 20% CV 20% CV
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 25
Staining, Destaining and DetectionStaining, Destaining and Detection
protein
micelles
destain
SDS + dye
detection
low background good signal to noise ratio SDS conc. below CMC
If no dilution was done the micelles would result in high background and low sensitivity
detection
X
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 26
Staining, Destaining and DetectionStaining, Destaining and Detection
protein
micelles
SDS + dye
destain
detection
low background good signal to noise ratio SDS conc. below CMC
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 27
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)
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 28
Clone Selection based on Protein Expression
1* 2 3 4 5 6 7 8*
Flu
ores
cenc
e
Time (seconds)
0
10
20
30
40
50
60
20 25 30 35 40 45 50 55
colony 1 colony 2
53.0
21.5
29.032.5
14.4
6.0
kDa
protein of interest
Example measured withProtein 50 kit
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 29
Monitoring of Protein Purification Process
2100 bioanalyzer: gel-like image Courtesy of P. Sebastian and S.R. SchmidtGPC-Biotech AG, Martinsried, Germany
cell lysate
wash(fraction 69)
elution (fraction 77)
flow through(fraction 66)
wash (fraction 78)
Time (seconds) 15 20 25 30 35 40 45
GFP Fusion Protein Analysis
2100 bioanalyzer: electropherogram
Example measured withProtein 200 plus kit
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 30
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).
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 31
Quality Control of Antibodies
90 kDa 160 kDa
intact antibody
16% half antibody
Determine the half antibody content in IgG preparations
Antibody analysis under reducing and non-reducing conditions
Ab reduced
Ab non-reduced
light chain
heavy chain
intact antibody
light + heavy chain
Ab reduce
d
Ab non-reduce
d
Absolute Quantitation of IgG samples
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 32
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
PBS blank runladder run
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 33
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 34
DNA Applications
mtDNA Screening
qPCR validation, impurity check
mPCR validation, impurity check
Restriction Digest
Analysis
Gene Expression
Oncology
Food Analysis
Clinical Research
ForensicTesting
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 35
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 36
marker
marker
25 bp
473, 500 bp
100, 105 bp
300, 315 bp
900, 1000 bp
2100 bioanalyzer dataGel-like image
25 bp
473, 500 bp
100, 105 bp
300, 315 bp
900, 1000 bp
2 % agarose gel stainedwith Ethidiumbromide
Data Format - Gel-Like Image c/w Agarose Gel
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 37
1 2 3 4 5
20001200
200
800
400
100
1 2 3 4 5
20001200
200
800400
100
Agilent 2100 bioanalyzer
Agarose gel
300 bp
3000 bp
Determination of PCR Product Impurity
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 38
Quantitative data from Agilent 2100 bioanalyzer
Sample c (DNA) main peak
300 bp PCR 41.4 ng/ul 40.7 ng/ul 300 bp PCR 1:4 9.6 ng/ul 9.6 ng/ul
Quantitative data from Agilent 2100 bioanalyzer
Sample c (DNA) main peak
300 bp PCR 41.4 ng/ul 40.7 ng/ul 300 bp PCR 1:4 9.6 ng/ul 9.6 ng/ul
1* 2 3 4 5 6 7*
Fluo
resc
ence
Time (seconds)
0
100
200
300
400
500
25 30 35 40 45 50 55 60 65 70 75 80 85 90
3000 bp PCR 3000 bp PCR 1:4 dil.
Impurity level : > 50%
3000 bp
Determination of PCR Product Impurity
1* 2 3 4*
Fluo
resc
ence
Time (seconds)
0
100
200
300
400
500
25 30 35 40 45 50 55 60 65 70 75 80 85 90
300 bp PCR 300 bp PCR 1:4 dil.
Impurity level : < 2%
300 bp
Quantitative data from Agilent 2100 bioanalyzer
Sample c (DNA) main peak
3000 bp PCR 61.9 ng/ul 40.7 ng/ul3000 bp PCR 1:4 14.8 ng/ul 9.8 ng/ul
Quantitative data from Agilent 2100 bioanalyzer
Sample c (DNA) main peak
3000 bp PCR 61.9 ng/ul 40.7 ng/ul3000 bp PCR 1:4 14.8 ng/ul 9.8 ng/ul
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 39
GMO Detection: Determination of GM Soya Percentage
Data kindly provided by CCFRA
Soya lectin gene target
EPSPS gene target: specific for Roundup Ready GM soya (Monsanto)
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 40
Effect of Multiplex PCR buffer on a 19-plex PCR
Data kindly provided by QIAGEN GmbH, Germany
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 41
Tumor Diagnostics
Data kindly provided by Adnagen
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 and DNA 500 LabChip kit
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 42
Detection of Single Base Mutations (1)in Exons 7 and 8 of the Human p53 Gene by RFLP Mapping using the DNA 7500 kit
Amplify exons 7 and 8 (resulting products: 618 bp fragment and 200 bp fragment)
Digest with Hpa II
Analyze using Agilent 2100 bioanalyzer and 4-20 % acrylamide gel
exon 7 exon 8
83 bp, 91 bp,168 bp, 276 bp
91 bp, 109 bpIn each example one of the restriction sites can be deleted by a point mutation
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 43
Detection of Single Base Mutations (2)
wt 106
200 bp109 bp
91 bp
91 bp91+83 bp
168 bp
wt 59
276 bp276 bp
251 bp
1*
2 3 4 5*
0
5
10
15
20
25
30
Flu
ore
sce
nce
Time (seconds)
30 35 40 45 50 55 60 65 70 75
p53Exon 7 wt/HpaII p53Exon 7 clone 59/HpaII
Lower Marker
Upper Marker267/268 bp
251 bp
166 bp
84/85 bp
1*
2 3 4*
0
5
10
15
20
25
Flu
ore
sce
nce
25 30 35 40 45 50 55 60 65
p53Exon 8 wt/HpaII p53Exon 8 clone 106/HpaII
Lower Marker
Upper Marker
Time (seconds)
208 bp111 bp
90 bp
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 44
Label free Analysis of Microsatellite InstabilitiesClinical 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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 45
Cell Applications
Protein Expression Monitoring
Intracellular
Transfection Efficiency Monitoring
Apoptosis Detection
Gene Silencing
Protein Expression Monitoring
Cell Surface Antibody Staining
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 46
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 47
Principle of Pressure-Driven FlowFor 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 micro-channels
Cells are hydrodynamically 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 micro-channels of the glass chip are filled with cell buffer
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 48
The Bioanalyzer Lab-on-a-Chip ApproachSeparation 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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 49
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
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 50
Flow Cytometry on a Chip - Two Color Detection- Three Types of Events
Blue/red cellsRed cells Blue cells
Dot plot view for easydata evaluation
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 51
Some Target applications
Apoptosis:
Annexin V Detection of phosphatidylserine on the cell surface
Caspase-3 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 procedureVerify silencing by cellular protein expression measurementCorrelation of siRNA uptake and gene knockdown
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 52
Typical workflow:
Cell assays: sample preparation
adherent
suspension(trypsinize) & harvest by centrifugation, wash
wash twice
Resuspend cells in cell buffer (LabChip Kit)
Load on chip
Add staining reagentand incubate
Data analysis to result
Customer Agilent
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 53
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)
Flow Cytometry on a Chip - Optics & Detection
Standard Flow Cytometers have 3-4 fluorescence detection channels:FL1: excitation 488 nm, detection 530 nmFL2: excitation 488 nm, detection 585 nmFL3: excitation 488 nm, detection 661 nmFL4: excitation 635 nm, detection 670 nm
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 54
Cell Assays - Applications: ApoptosisAnnexin Binding
Phosphatidyl-serine from inner leaflet flips to outer membrane Phosphatidyl-serine from inner leaflet flips to outer membrane during apoptosis and can be labeled by Annexin Vduring apoptosis and can be labeled by Annexin V
Healthy cellHealthy cell
““Live” apoptotic cellLive” apoptotic cell
Dead cellDead cell
Live dye: Calceinbiotin-Annexin+ Cy5-streptavidin
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 55
Annexin V Assay (24h Induction)
0%
20%
40%
60%
80%
0 10 20 30
Sample Number
% A
po
pto
tic
2100-1
2100-2
2100-3Flow cytometer
5 chips, each loaded with control in well 1 and 24h sample in wells 2-6
Three Bioanalyzer instruments vs a flow cytometer reference instrument
Chip 1 Chip 2 Chip 3 Chip 4 Chip 5
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 56
Applications: Protein Expression AnalysisGFP Transfection Efficiency Control
Disclaimer. Neither Agilent nor Caliper makes any representation that use of EGFP andpractice of the methods described in this publication is licensed orotherwise authorized under any third party patents, including patents ownedor exclusively licensed by Aurora Biosciences or Amersham Biosciences.
CHO-K1 cells were transfected with EGFP DNA and Lipofectamine.
GFP transfected cells
Mock transfected cells
0.1 %
56.6 %
Control
EGFP transfected
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 57
GFP Transfection Efficiency
0%
10%
20%
30%
40%
50%
60%
70%
1 2 3 4 5
Chip Number
Tra
ns
fec
tio
n E
ffic
ien
cy
Flow cyt.
2100-1
2100-2
2100-3
2100-1 2100-2 2100-3 All Flow cyt.ctrl mean 0.46 0.31 0.47 0.40 0.16
SD 0.08 0.29 0.43 0.29 0.12
GFP mean 60.19 59.15 59.26 59.53 60.90SD 2.13 2.48 2.10 2.26 1.22
%CV 3.54 4.19 3.54 3.80 2.01
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 58
Flow Cytometry Assays Applications - Cell surface Antibody staining
Cell expressingprotein of interest
Target protein
Live dye: CalceinCy5 or APC-labeled Antibody
Cell not expressingprotein of interest
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 59
Extracellular Antibody Staining
Averaged data per instrument
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
1 2 3 4 5 6
sample #
% g
ated
% gated 2100-1
% gated 2100-2
% gated 2100-3
%gated 2100-4
Flow cytometer
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
Mean % CD3+ cells2100-1 2100-2 2100-3 2100-4 Flow cyt.60.9 67.8 66.6 65.0 60.934.4 36.7 36.7 34.3 29.817.3 17.6 18.7 17.2 13.88.9 9.4 9.9 8.3 6.55.1 4.4 5.3 4.9 3.20.8 0.6 0.3 0.3 0.0
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 60
15 min
cells + CBNF
15 minOn-chip
Spin, aspirate,resuspend,spin, aspirate
CBNF
10 min 10 min 10 min15 min
35 minConventional
Resuspend cells in CB
GFP On-Chip Staining - Workflow
Group/Presentation TitleAgilent Restricted Month ##, 200XPage 61
GFP On-Chip Staining - Histogram Quality
2100 bioanalyzer
Flow Cytometer