Joseph B. Martin Conference Center, Boston, MA Cover of Assay … · 2019-05-02 · High Content...
Transcript of Joseph B. Martin Conference Center, Boston, MA Cover of Assay … · 2019-05-02 · High Content...
High Content 2017September 13th-15th4th Annual Conference
San Diego Conference Center, San Diego, CA
Assay Development
Considerations for HCSSource: NIH/NCATS
Assay Guidance Guidelines
Joe TraskSenior Application Scientist
PerkinElmer, Inc.
email: [email protected]
High Content 2018September 18th-20th
5th Annual Conference
Joseph B. Martin Conference Center, Boston, MA
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
WHY ASSAY DEVELOPMENT ???
http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970
SEE VIDEO !!!
1,500 scientists lift the lid on reproducibility,
Nature Survey May 2016
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
WHY ASSAY DEVELOPMENT ???
http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970
SEE VIDEO !!!
1,500 scientists lift the lid on reproducibility,
Nature Survey May 2016
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
WHAT IS “HIGH CONTENT” IMAGING?
High Content screening is an automated cell biology method drawing on optics, chemistry, biology and image analysis to permit rapid, highly parallel biological research and drug discovery. (Source: Wiki)
“High Content” in the context of image
analysis provides a few to hundreds of
measureable “features” per cell or object
being interrogated without multiplexing.
Multiplexing adds to the overall value of
the data but it is not high content on its
own.
HC Features – these are measureable
statistical pieces of information of
structures within the cell. A simple
structure are spots or edges; complex
structures are considered objects
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HIGH CONTENT _________?
HCS – High Content Screening
HCA – High Content Analysis
HCI – High Content Imaging
IC – Image Cytometry
Computer-assisted automated microscopy
Computer-assisted image analysis
THE IMAGE IS DATA…THE GOOD, BAD, & UGLY
Cytotoxic Fluorescent
Media Anisomycin
Size
Shape
Intensity
Texture
Dynamics
Distribution
Multiparametric
Multivariate
Rarely
Homogeneous
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
IMAGE PROCESSING OVERVIEW
Figure adopted by Defineins, IncSource: Adopted from Definiens
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
ASKING THE RIGHT QUESTIONS…
• What is an HCS assay going to provide me that other assays do not?
• Will it provide more relevant information than other assays?
• Does the data outputs provide not only a result but also locate the target’s activity in the cell (location of a phosphorylated protein)
• Can phenotypic or target-based measurements including morphology be quantified?
Question that should be asked:
Same label, different distributionLocation,
Location,
Location.
Modified from presentation at the RNAi Global Initiative Teleconference - HCA Assay Development, 17-Sep-2009, OJ Trask
pH3-AF647, captured on Operetta CLS, 63xW
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCS EXPERIMENTAL WORKFLOW
Seed Treat StainGrowCells
Cell models
Compounds
Multi-well
plates
AntibodiesFluorescent
Dyes
Wetware
siRNAs
CRISPR
Chemistry
AnalyzeImage
HCS
Hardware
Liquid Handling
Robotics
Data analysis
Knowledge
High Content ProfilerTM
Software
StatisticsData
Management
MachineLearning
Slide courtesy of PerkinElmer
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU
NEED TO KNOW…
https://www.ncbi.nlm.nih.gov/books/NBK100913/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
PROBING CELL HEALTH & STRESS INDICATORS
Presented at SLAS, 2014, The Hamner Institute, J. Trask
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU
NEED TO KNOW…
https://www.ncbi.nlm.nih.gov/books/NBK100913/
14 Cellular Imaging & Analysis Webinar Series2017-May-24
Advanced & Complex Cell Models
Demand for better in vitro models to recapitulate the in vivo environment
Modelcells, tissue, organisms
Alternative Animal Studies Develop
Biology
Cancer Biology
Toxicology
Neurobiology
Drug Discovery / Pre-clinical
Immunology
Metabolic Diseases
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CELL MODEL CHOICEIS IT REPRESENTATIVE OF BIOLOGY OR PHENOTYPE?
Example in Toxicology: Human in vitro Liver Cell Models
Model Source
Hep G2 Hepatocellular carcinoma, epithelial, 15yo caucasian male; Wistar
Institute, 1980.
HuH 7 Differentiated hepatocyte derived cellular carcinoma cell line
originally taken from a liver tumor in a 57yo Japanese male in 1982
THLE-2 Derived from primary normal adult liver, left lobe, by infection with
SV40 large T antigen. NIH, 1993.
HepaRG™ terminally differentiated hepatic cells derived from a human hepatic progenitor
cell line that retains many characteristics of primary human hepatocytes.
Stem cells: (1) ES and (2) iPS derived
Primary, cryopreserved
Primary, freshly isolated – Hu difficult to get
Coculture systems, e.g., hepatocytes with stellate, kupffer, and stroma cells
Tissue
Co
mp
lex
ity
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
Types
• Immortalized Cell Lines
• Stem Cells
• Primary Cells
• Explant Tissue
• Organisms
Characteristics
• What is the source
• What background information is available
• Genotype / Phenotype
• Engineered
• Mixed coculture
• Adherent or suspension
• 2D, 3D, 4D (t)?
Are the cells conducive to imaging?
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CHOICE OF CELL LINE
Resources• Literature references
(http://www.Pubmed.com)
• Existing protocols
(http://www.currentprotocols.com/WileyCDA)
• American Type Culture Collection (ATCC) (http://www.atcc.org)
• Stem Cell Consortium at NIH CRM https://commonfund.nih.gov/stemcells/lines
• Scientific Societies & Journals
• Other commercial sources
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HELA CELL LINEIS IT EVERYWHERE?
• 1951, Henrietta Lacks (HeLa), a patient who died of her cancer.
• The cells from Lacks' cancerous cervical tumor were taken without her knowledge or consent by researcher George Gey.
• 1952, HeLa, the first human cells grown in a lab that were naturally “immortal”
• 1954, Jonas Salk developed a vaccine for polio using these cells
• 1955, HeLa cells were the first human cells successfully cloned
• 1967, Stanley Gartler & 1975, Walter Nelson-Rees were the first to publish on the contamination of various cell lines by HeLa
• Scientists have grown an estimated 20 tons of her cells
• Approximately 11,000 patents involving HeLa cells
• HeLa contaminated Cell Lines
• Why does ATCC continue to distribute HeLa Contaminated Cell Lines? ATCC continues to distribute these cell lines, even though they have been shown to be contaminated with HeLa, because researchers need them for purposes beyond use as models for specific disease/original source tissue. For example, ATCC® CCL-17 ™, KB cells are used as a model for folate receptors; ATCC® CCL-23 ™, HEp-2 cells are used as a host to grow Chlamydia and RSV. Source: https://www.atcc.org/Global/FAQs/3/6/HeLa%20contaminated%20Cell%20Lines-1207.aspx
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CELL LINECELL DENSITY EFFECTS ON BIOLOGY & WINDOW
Lower Cell Seeding Density Higher Cell Seeding Density
Neurite Outgrowth
Receptor Internalization
Migration / Tracking
WoundHealing
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
ENGINEERED CELL LINE
Making your own constructs and cell lines:
Requires molecular biology expertise
transient transfections, stable transfections, or retroviral
Requires flow cytometry or other techniques to generate clonal populations
Typically easy to culture, may need to maintain under selection
Usually maintain viability and phenotype over several passages
Need to prove the biology works
MK2-EGFP RetrovirusConstruct
Untreated
Anisomycin / TNF-a
REF: Trask OJ , et al., Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-
activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform. Methods Enzymol. 2006;414:419-39.
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CHOICE OF CELL LINE
HCS Assay with Non-adherent Cells
Jurkat cells plated on fibronectin coated 96-well platesStained with Hoechst (left) and Phalloidin (right)
U937 cells plated on
96 well LiveCell
Array microplate
20um diameter picowells
(left) Hoechst stained (right)
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CHOICE OF CELL LINE…THE RIGHT STUFF
Cell growth conditions • Each cell type / cell model has specific medium optimized for growth
• Serum type is usually specified with recommended medium type
• Some cells may require Horse serum or other sera in addition to FBS
• Serum is almost always required for growth conditions unless the medium has been optimized for serum free conditions
• Typical serum range is 0-20%
• Additional growth components may be required
• Conditioned medium may be needed for primary cells
• Cell treatment options• Medium used during the treatment may differ from growth media
• Serum type and concentration may vary in assay conditions with low or serum free conditions used for compound testing (to remove effects of serum)
• Incubation times for treatment of stimulants and or compounds are dependent on the response desired and biological relevance
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CELL CULTURE HIGH PASSAGE LEADS TO SIGNAL LOSS
Limit extended passage of cells
Maintain the quality & reproducibility of your HCS assay
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CELL CULTURE PASSAGE OF CELLS AFFECTS SIGNAL
ER
K-1
/2 C
yto
Nu
cD
iff
ƞg/ml PMA
Fo
ld W
ind
ow
p13 p28 p40
Work done by Debby Nickischer, 2002-04, Sphinx Laboratories, Eli Lilly
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CELL CULTURE PASSAGE OF CELLS AFFECTS SIGNAL
Cell Passage Number Comparison. Different cell splitting passages of HeLa cells seeded at 5,000 cells/well overnight
and treated with dose response of TNF-α for ~35 minutes, then fixed and stained to measure NF-κB translocation. Plates
were analyzed on HCS imager to determine NF-κB translocation using CytoNuc Difference calculation; data was
normalized to control and plotted in GraphPad Prism using non-linear regression 3-parameter fit.
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CELL CULTURE IMPACT OF SPLITTING CELLS ON DIFFERENT DAYS
Source: Presented at 2001 IBC High Content Imaging Workshop, Bal Harbor, FL, J. Trask
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
CELL SEEDING DENSITYCELL DENSITY AFFECTS ASSAY WINDOW
AND IMAGE ANALYSIS ALGORITHM PERFORMANCE
REF: https://www.ncbi.nlm.nih.gov/books/NBK100914/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU
NEED TO KNOW…
https://www.ncbi.nlm.nih.gov/books/NBK100913/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
BEWARE: NOT ALL MICROPLATES ARE EQUAL
Polymer Based Glass
→ANSI/SBS/SLAS Standard
→ Not all wells have same surface area
or volume capacities
Biggest differences:
• Bottom thickness
• Bottom thickness variability
• Skirt height
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
BEWARE: REVIEW IMAGE WELLS FOR FOCUS
Unevenness Across the Well Image Quality: The Affects on Algorithm
Reference: Paul A. Johnston and Oscar J. Trask (2017 – in press) Methods in Molecular Biology Series: High
Content Screening A Powerful Approach to Systems Cell Biology and Drug Discover. ISBN 978-1-4939-7355-2
Objects Ident =35 Objects Ident =20
Match objective lens with the microplate thickness!384w microplate
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
MICROPLATE COATINGAFFECTS BIOLOGICAL OUTCOME
Source: Presented at 2001 IBC High Content Imaging Workshop, Bal Harbor, FL, J. Trask
Source: Presented at 2001 IBC High Content Imaging Workshop, Bal Harbor, FL, J. Trask
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
MICROPLATE COATINGAFFECTS BIOLOGICAL OUTCOME
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
Source:
BD website
MICROPLATE COATINGAFFECTS BIOLOGICAL OUTCOME
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
3D microplate formats-Most are composed of Matrigel, Hydrogel, Agrose,
ECM or other substrate
-Take hours to days to form spheroids
-Not all cells form spheroids
Types
• Suspension or “hanging drop”
• Ultra low attachment
• Magnetic Beads
• Biomaterials/films
• Others?
Which Model: ULA Microplates & Spheroid Phenotypes
Imaging
Cell Models: InSphero GravityPLUSTM
HCT116
Opera 20xW, z-stack maximum intensity
projection. Hoechst, GFP, RFP
NIH3T3
Imaging
20x
100 µm
Cell SeedingMicrotissue
Maturation
Microtissue
Transfer
Microtissue
Assay
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU
NEED TO KNOW…
https://www.ncbi.nlm.nih.gov/books/NBK100913/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
Verify your probe choice is detectable with instrument
• Resolution required & wavelength
SBI2 Educational Course, Steve Haney
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
Source: 2016 SBI2 Educational Course, Steve Haney
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
TYPICAL HCS PLATE PREPARATION -FIXATION
• Test with each biology and cell model
• Type of fix
• Age / stability
• Amount (%)
• Pre-warm to 37oC
FixationFix 30 min, Perm 15 min
0
50
100
150
200
250
300
350
1 2
avg
cyto
nu
c d
iff .9
1
1.8
2
3.7
4
% fix
Formaldehyde
Untreated Treated
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
TYPICAL HCS PLATE PREPARATION –FIXATION PERMEABILIZATION
• Permeabilization
• Triton X-100 or other detergent
• Permeabilizing fixed cell membranes
• allows antibodies and dyes access
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
REAGENT EVALUATION& DETERMINATION
Antibody Titration Experiment
Graph of commercially available antibodies evaluated
X-axis represents 1oAb concentration with 10μg/ml 2oAb;
Y-axis represents average fluorescent intensity of NF-κB difference between the cytoplasm and nucleus
subtracted from the 2oAb control. Values listed below x-axis are the raw data (CytoNuc Diff).
Considerations
• Establish background /non-
specific binding of 2oAb
• Maintain 2oAb Conc during 1o
Ab titration
• If multiplexing, test cross-talk
of fluorescent spectra & non-
specific binding of other
antibodies or probes.
• Fixation & permeablization
methods are not standardized
___proteins may require further
optimization.
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
REAGENT STABILITY LOT-TO-LOT & FREEZE-THAW CYCLES
Stability of cytokines following multiple freeze-thaw
cycles. Following the reconstitution of the cytokine per manufacture
suggestion, cytokine reagents were store at –80oC, and then allowed
to thaw at room temperature before use. Samples were then re-frozen
at –80oC multiple times. Translocation of NF-kB was performed on
HeLa cells following treatment with IL-1α (left) or TNF-α (right) as
previously described and data was normalized to control and plotted
in GraphPad Prism using non-linear regression 3-parameter fit.
REF: https://www.ncbi.nlm.nih.gov/books/NBK100914/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
PROBE STABILITY POST FIXATION
Stability of NF-kB-p65-AF488 complex post staining and fixation. Using several plates, HeLa cells seeded
at 5,000 cells/well overnight and treated with 25ηg/ml of TNF-α for ~35 minutes, then fixed and stained to
measure NF-κB translocation at the maximum signal. At different time points (days), plates were analyzed
on HCS imager to determine NF-κB translocation fluorescent intensity measurements using CytoNuc
Difference calculation; raw data was used and plotted for comparison.
REF: https://www.ncbi.nlm.nih.gov/books/NBK100914/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU
NEED TO KNOW…
https://www.ncbi.nlm.nih.gov/books/NBK100913/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
The liquid plumr effect – at high concentrations of DMSO• Can cause inconsistent effect on cells in areas of well• Can kill cells
Ways to solve• Pre-dilution in media• Mixing during addition
Cells don’t receive compound
Cells receive too much compound
High Concentration DMSO Low Concentration DMSO
Compound Distribution among cells more even
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
DMSO TOLERANCE
REF: https://www.ncbi.nlm.nih.gov/books/NBK100914/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
COMPOUND SELECTION IMPLEMENTATION OF CONTROLS
• Known inhibitors – Biology Control
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
COMPOUND SELECTION CHOICE OF STIMULI
Stimulant Concentration CurvesErk Cyto-Nuc Activation
0.000000010.00000010.0000010.00001 0.0001 0.001 0.01 0.1 1 10 100 1000
50
100
150
200
250
300
350
400
450
500
550 OSM
PMA
TGF
EGF
TNF
[Log] ng/ml
Mean
Cyto
Nu
cD
iff
R²
OSM
0.9731
PMA
0.9197
TGF0.9717
EGF
0.9016
TNF0.7092
EC50
OSM
0.1852
PMA
1.204
TGF0.003484
EGF
0.4793
TNF0.03396
Work done by Debby Nickischer, 2002-04, Sphinx Laboratories, Eli Lilly
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
COMPOUND SELECTION CHOICE OF STIMULI
Activation of NF-κB-p65 with Different Stimuli. HeLa cells seeded at 5,000 cells/well overnight and treated with
stimuli for 30 minutes, then fixed and labeled with NF-κB-p65-AF488 and Hoechst33342 to measure NF-κB
translocation. Plates were analyzed on HCS imager to determine NF-κB translocation using CytoNuc Difference
calculation; data expressed as raw unit values (y-axis) from algorithm using non-linear regression 3-parameter fit
was done in GraphPad Prism; standard deviation error bars (n=3) was removed for visualization.
REF: https://www.ncbi.nlm.nih.gov/books/NBK100914/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU
NEED TO KNOW…
https://www.ncbi.nlm.nih.gov/books/NBK100913/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
When using secondary treatment / stimuli – things to ask?
• Pre-treat with compound
• Simultaneous addition of all test articles
• Are treatment times amenable for screening
Long Incubations
• Bolus treatment
• Remove ALL and add ALL compounds/medium
• Remove ½ volume and add ½ volume compounds/medium
Frames per second
HeLa-MK2-EGFP cells, 1 frame/ 2min for 64 min
TNFɑ added frame 1 (time 0), Compressed run time 25 secs
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
KINETICS… DETERMINING A ROBUST STIMULI RESPONSE
pE
RK
-1/2
Work done by Debby Nickischer, 2002-04, Sphinx Laboratories, Eli Lilly
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
KINETICS… DETERMINING A ROBUST STIMULI RESPONSE
NF-κB Translocation time course kinetics. HeLa cells seeded at 5,000 cells/well overnight and treated
with 25ηg/ml of IL-1α over time, at 5 or 10 time minute intervals, cells were fixed and then stained to
measure NF-κB translocation. Plates were analyzed on HCS imager to determine NF-κB
translocation using CytoNuc Difference calculation; data was normalized and plotted in GraphPad
Prism using non-linear regression one-site binding to calculate the ½ time response, 24 minutes.
REF: https://www.ncbi.nlm.nih.gov/books/NBK100914/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCI ASSAY DEVELOPMENTBASIC PRINCIPLES & THINGS YOU
NEED TO KNOW…
https://www.ncbi.nlm.nih.gov/books/NBK100913/
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCS STATISTICAL MEASUREMENTSHOW MANY FIELDS PER WELL TO
DETERMINE STATSISTICAL SIGNIFCANCE?
REF: Trask O. Joseph Jr., Moore Amanda, and LeCluyse Edward L., A Micropatterned Hepatocyte
Coculture Model for Assessment of Liver Toxicity Using High-Content Imaging Analysis, ASSAY and
Drug Development Technologies 2014 12 1 , 16 -27
*
**
***
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
HCS STATISTICAL MEASUREMENTS3D ASSAY OPTIMIZATION Z’-FACTOR
-0.22
0.210.17
0.53
0.67
60µm
1
-
92%
2
60
85%
3
30
77%
5
15
62%
13
5
0
Ref: Curiosity of PerkinElmer and InSphero
Number of Planes
Distance, µm
Decrease in Acquisition
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
• Review of information for final conditions
• Cell model; microplate type & coating
• Cell Seeding density & medium conditions
• Optimal stimuli and/or reference compound & dose
• Optimal time of stimulus and/or treatment
• Optimal staining conditions; live, fixed, stability
• Validation - 3 day or 3 independent experiential IC/EC50
• Determine repeatability and reproducibility of the assay
• Well to well variance measurements by:
• Full plate of untreated/DMSO/Stimuli and compound
• ½ plate of each by columns/rows or checkerboard
• Reference library of compounds @ defined dose
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
ST
IMU
(%)
-20
020
4060
8010
012
0
1 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%
Book:Page= A04267:022 Screen: BCATENINTRANSLOC RAPID80Plates: 1 to 20 Overlay plots
A B C D E F G H
ST
IMU
(%)
-20
020
4060
8010
012
0
ST
IMU
(%)
-20
020
4060
8010
012
0
1 12 24 36 48 60 72 84 961 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%
Book:Page= A04267:022 Screen: BCATENINTRANSLOC RAPID80Plates: 1 to 20 Overlay plots
A B C D E F G H
Hit Cytotoxic Compound Fluorescent Compound
Ref: Borchert K, et al., High-content screening assay for activators of the Wnt/Fzd
pathway in primary human cells. Assay Drug Dev Technol. 2005 Apr;3(2):133-41.
IMAGE ARTIFACTSCYTOTOXICITY & FLUORESCENT
September 18, 2018
OJ TraskAssay Development Educational Course
5h Annual Conference, Boston, MA
ST
IMU
(%)
-20
020
4060
8010
012
0
1 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%
Book:Page= A04267:022 Screen: BCATENINTRANSLOC RAPID80Plates: 1 to 20 Overlay plots
A B C D E F G H
ST
IMU
(%)
-20
020
4060
8010
012
0
ST
IMU
(%)
-20
020
4060
8010
012
0
1 12 24 36 48 60 72 84 961 12 24 36 48 60 72 84 96mTOP=83.083 mSTD=-247.4 mBKGD=2.0905 m Cont50=165.0 tHits=19 mTopCV=12.0% mBotCV=7.9%
Book:Page= A04267:022 Screen: BCATENINTRANSLOC RAPID80Plates: 1 to 20 Overlay plots
A B C D E F G H
Hit Cytotoxic Compound Fluorescent Compound
Review Images!!!
Ref: Borchert K, et al., High-content screening assay for activators of the Wnt/Fzd
pathway in primary human cells. Assay Drug Dev Technol. 2005 Apr;3(2):133-41.
IMAGE ARTIFACTSCYTOTOXICITY & FLUORESCENT
Assay Development for
High Content Screening August 07, 2017
OJ TraskNIH/NCATS Assay Guidance Workshop for High Throughput Screening and Lead Discovery
William F. Bolger Center, Potomac, Maryland
1. Assay Development Guidelines for Image-Based High Content Screening, High
Content Analysis and High Content Imaging. William Buchser, Mark Collins, Tina
Garyantes, Rajarshi Guha, Steven Haney, Vance Lemmon, Zhuyin Li, and O. Joseph Trask.
2. Advanced Assay Development Guidelines for Image-Based High Content
Screening and Analysis. Mark-Anthony Bray, Anne Carpenter, and Imaging Platform, Broad
Institute of MIT and Harvard.
3. Nuclear Factor Kappa B (NF-κB) Translocation Assay Development and
Validation for High Content Screening. O. Joseph Trask Jr.
4. High Content Screening with Primary Neurons. Hassan Al-Ali, Murray Blackmore, John L
Bixby, and Vance P. Lemmon.
5. New chapters under consideration
• Fluorescent artifacts – Q4 (authors: Steve Haney, Paul Johnston, Steve Titus, Joe Trask)
• 3D imaging
• Machine learning and analytics
• Toxicology
• Others…
NIH/NCATS ASSAY GUIDANCE MANUAL HCI CHAPTERS
https://www.ncbi.nlm.nih.gov/books/NBK53196/
NIH / NCATS Assay Guidance Manual
Assay Development for High Content Screening & Best Practices for 3D HCS
September 18, 2018
OJ Trask
RESOURCES
https://www.ncbi.nlm.nih.gov/books/NBK53196/
www.sbi2.org [email protected]
High Content Screening (2018). A Powerful Approach to
Systems Cell Biology and Phenotypic Drug Discovery
Editors: Johnston, Paul A., Trask, Oscar J. (Eds.)
ISBN 978-1-4939-7357-6
• “3D High-Content Screening of Organoids for Drug Discovery”, Dan LaBarbera, Ph.D.,
Univ of Colorado
• “HCS for predictive toxicology and in vitro pathology using simple 3D microtissues”,
Kim Boekelheide, M.D., Ph.D., Brown University
• “Face-to-face with cancer: The power of high-content imaging and analytics to illuminate
cell population dynamics”, Shannon Mumenthaler, Ph.D., Univ of Southern CA.