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CellCiphrTM Cytotoxicity HCS Profiling Panel,Human HepG2 Cells
Andrew J. Ball*, Kenneth A. Giuliano†, William Irwin†, Janet L. Cuy*, Patricia A. Johnston†, Theresa L. Schaub*, Rick L. Ryan*, Dennis W. Harris*, Patrick A. Schneider, D. Lansing Taylor† & Matthew Hsu*
*Millipore Corporation, Bioscience Division, 28820 Single Oak Drive, Temecula, CA 92590. Email: [email protected]
†Cellumen, Inc., 2516 Jane Street, Pittsburgh, PA 15203
Copyright © 2007 Millipore Corporation
AbstractDrug-induced hepatotoxicity is a major factor in the high fail rate of drug development, and in withdrawal of drugs from the market. High-Content Screening
for measurement of effects of lead compounds on multiple cellular pathways is a valuable tool for drug discovery. Millipore and Cellumen have developed the CellCiphrTM cytotoxicity profiling assay based on Cellumen’s Cellular Systems Biology approach, and a kit comprised of high-quality, validated, multiplexed detection reagents and protocols for profiling multiple human hepatotoxicity endpoints in HepG2 cells. 11 cytotoxicity parameters are measured in HepG2 cells over 3 time points – acute (1 hr), early (24 hr) and chronic (72 hr). Parameters measured are: 1. Cell Loss, 2. Cell Cycle Arrest, 3. DNA Degradation/Apoptosis, 4. Nuclear Size, 5. Oxidative Stress, 6. Stress Kinase Activation, 7. DNA Damage response, 8. Mitochondrial Membrane Potential, 9. Mitochondrial Mass, 10. Mitotic
Arrest, 11. Cytoskeletal Integrity. The assay is performed in collagen-coated 384 well microplates, with two plates utilized for each timepoint. Reagents and materials supplied in the Millipore (co-branded with Cellumen) CellCiphrTM
Cytotoxicity Kit are platform independent and sufficient for detection of all 11 parameters at 3 time points for 4 control toxins (anisomycin, camptothecin, CCCP, paclitaxel) and 4 to 16 unknown compounds, each in two 10-point dose response curves. The kit includes buffers for Compound Dilution, Cell Fixation and Permeabilization, Antibody Dilution and Washing. All reagents and protocols have been validated for analysis on both the GE IN Cell Analyzer 1000 and the Cellomics ArrayScan. Our data demonstrates the effectiveness of the CellCiphrTM
Cytotoxicity kit in detection of each hepatotoxicity parameter in response to a range of doses of the 4 control toxins. The combination of unique antibody pairs and detection reagents allows detection and analysis of the cellular systemic response to toxin challenge.
Millipore and Drug Discovery
High Content Screening (HCS) technology represents a major step towards improving the drug discovery process. HCS enables the evaluation of multiple biochemical and morphological parameters in cellular systems. Recent data indicate that multiparametric High-Content Screening for cytotoxicity using human HepG2 cells has ahigh concordance with drug-induced human hepatotoxicity (O’Brien et al., 2006). Successful HCS assays rely on high-quality detection reagents. Accordingly, Millipore, in collaboration with Cellumen, Inc., have developed the CellCiphr™ Cytotoxicity Profiling Panel for human HepG2 cells, a multiplexed High Content Screening kit comprising high-quality, validated, compatible detection reagents and validated protocols for profiling 11 human hepatotoxicity end-points in two 384 well microplates (Table 1, Fig 5). The CellCiphr™Cytotoxicity Profiling Panel for human HepG2 cells provides a means to screen compounds for a broad range of potentially toxiceffects early in the drug discovery process, and provide better information to drive drug development. The assay allows detection of 11 parameters at three separate time points (e.g. acute – 30 min, early – 24 hr, chronic – 72 hr). Reagents and materials supplied in the HCS100 CellCiphr™ Cytotoxicity Kit are sufficient to perform ten point dose response curves in duplicate for four control toxins (provided) and sixteen unknown compounds at each time point.
Introduction
Related Products from Millipore
Summary
HCS100 Assay Kit Components
HCS200 Neurite Outgrowth Assay for HCS
1. Antibody A, 200x. Oxidative Stress Marker: 1 vial containing 100 µL.
2. Antibody B, 200x. Anti-Antibody A, FITC conjugate: 1 vial containing 100 µL.
3. Antibody C, 200x. Stress Pathway Marker: 1 vial containing 100 µL.
4. Antibody D, 200x. Anti-Antibody C, Cy3 conjugate: 1 vial containing 100 µL.
5. Antibody E, 200x. DNA Damage Marker: 1 vial containing 100 µL.
6. Antibody F, 200x. Anti-Antibody E, Cy5 conjugate: 1 vial containing 100 µL.
7. Antibody G, 200x. Mitotic Arrest Marker: 1 vial containing 100 µL.
8. Antibody H, 200x. Anti-Antibody G, FITC conjugate: 1 vial containing 100 µL.
9. Antibody I, 200x. Microtubule Stability Marker: 1 vial containing 100 µL.
10. Antibody J, 200x. Anti-Antibody I, Cy5 conjugate: 1 vial containing 100 µL.
11. Mitochondrial function marker, 20,000x: 1 vial containing 15 µL.
12. Nuclear Stain Solution, 200x: 1 vial containing 200 µL.
13. Compound Dilution Buffer: 1 bottle containing 500 mL.
14. Cell Fixation Buffer: 1 bottle containing 200 mL.
15. Cell Permeabilization Buffer: 1 bottle containing 100 mL.
16. Antibody Dilution Buffer: 1 bottle containing 100 mL.
17. Wash Buffer: 2 bottles each containing 1000 mL.
18. Control Toxin 1, Anisomycin, 2.5 mM in DMSO, 250x: 1 vial containing 100 µL.
19. Control Toxin 2, Camptothecin, 2.5 mM in DMSO, 250x: 1 vial containing 100 µL.
20. Control Toxin 3, CCCP, 25 mM in DMSO, 250x: 1 vial containing 100 µL.
21. Control Toxin 4, Paclitaxel, 0.25 mM in DMSO, 250x: 1 vial containing 100 µL.
22. 20 gauge needle with syringe, 5cc capacity: 6 each, sterile, disposable.
23. Plate Sealers: 6 each.
References
O'Brien PJ, Irwin W, Diaz D et al. Arch Toxicol. 2006 80:580-604.
Giuliano KA, Johnston PA, Gough A, Taylor DL. Methods Enzymol.2006 414:601-619.
Giuliano KA. Methods Mol Biol. 2007 356:189-193.
CellCiphr™ Assay Design
Typical HCS Workflow
CellCiphr™ Assay Overview
1. Plate HepG2 cells on 384 well microplates. Incubate for 18 hr.
2. Treat cells with control toxins (below) and test compounds.
i. Camptothecin: 10 point dose response, Maximum final conc. = 10 µM
ii. Anisomycin: 10 point dose response, Maximum final conc. = 10 µM
iii. CCCP: 10 point dose response, Maximum final conc. = 100 µM
iv. Paclitaxel: 10 point dose response, Maximum final conc. = 1 µM
3. Label cells with physiological indicator dyes, primary antibodies and corresponding secondary antibodies.
4. Perform HCS imaging and analysis.
5. Data Interpretation - obtain Human Cytotoxicity Profile
HCS150 ECM-coated 384 well plates for CellCiphr™ assay
� Screen compounds for a broad range of potentially toxic effects early in the drug development process.
� Accelerate prioritization of lead compounds.
� Gain insights into mechanism(s) underlying drug actions.
� Assay utilizes a Cellular Systems Biology approach.
� Relevant to human cytotoxicities.
� Multiplexed HCS analysis of eleven human cytotoxicity parameters using two 384 well microplates.
� Detection of eleven human cytotoxicity parameters over up to 3 time points.
� Generate ten-point dose response curves in duplicate for up to 16 test compounds and 4 control toxins (provided).
� Proprietary panel of high quality detection reagents provides high sensitivity and selectivity.
� Optimized HepG2 cell handling protocol enables consistent cell plating.
� Fully leverages High Content Screening.
Results
Figure 2. Examples of cytotoxicity profiles generated using the
CellCiphr™ Cytotoxicity Profile, Human HepG2 Cells.
Data demonstrating the determination of IC50 values of a range of
toxins upon parameters such as cell count, nuclear area, cell cycle,
oxidative stress, stress kinase activation, DNA damage, mitochondrial
membrane potential, mitochondrial mass, mitotic arrest, and
microtubule stability in response to following 24 hr exposure to each
compound is shown. Cell handling was performed as recommended
in the HCS100 CellCiphr™ assay protocol; detection reagents and
protocols used were as provided with the HCS100 CellCiphr™ assay
kit.
A&B. Effect of anisomycin on stress pathway activation. Untreated cells have some nuclear labeling
(A) that becomes more intense with 40 µM anisomycin treatment (B). C&D. Effect of CCCP on
mitochondrial function. Untreated cell has well defined mitochondria (C), cells treated with 126 µM
CCCP have less intense, more diffuse mitochondrial labeling (D). E&F. Effect of paclitaxel on
cytoskeletal integrity/microtubule stability. Untreated cell has an extensive array of single
microtubules (E), cell treated with 20 µM paclitaxel has stabilized microtubules with evidence of
bundling (F). G&H. Effect of camptothecin on DNA damage response. Untreated cells have low
levels of DNA damage marker (G), cells treated with 32 µM camptothecin exhibit activated nuclear
DNA damage marker (H). I&J. Effect of camptothecin on oxidative stress. Untreated cells maintain
low levels of oxidative stress marker (I), cells treated with 100 µM camptothecin exhibit elevated
levels of marker within their nuclei (J). K&L. Effect of paclitaxel on mitotic arrest. Untreated cycling
cells exhibit mitotic arrest marker only during normal mitoses (I), cells treated with 6.3 µM paclitaxel
show abnormal mitoses with elevated mitotic arrest marker (J). M&N. DNA degradation marker and
20 µM paclitaxel. Untreated cells show normal nuclear morphology (M), subpopulations of cells
treated with 20 µM paclitaxel exhibit fragmented nuclei (N). O&P. Image sets of control and 0.2 µM
paclitaxel treated (24 hr) HepG2 cells. Blue-Nuclei; Green–Microtubules; Red–Mitochondria;
Magenta–Mitotic Arrest marker. Untreated cells show a well defined microtubule cytoskeleton and
normal mitochondria and nuclei. The image also includes one cell undergoing mitosis (O). When
treated with 0.2 µM paclitaxel, subpopulations of cells exhibit bundled microtubules, fragmented
nuclei, and elevated mitotic arrest marker levels (P). Cell handling protocols, detection reagents and
protocols used were as provided with the HCS100 CellCiphr™ assay kit.
Figure 3. Assay is
platform-independent.
Comparison of data
from plates analyzed on
Cellomics ArrayScan
VTI (upper panels) vs
GE IN Cell Analyzer
1000 (lower panels).
Plate Layouts
Table 1. Cell parameters measured by
CellCiphr™ Cytotoxicity Profiling Panel
Figure 5. Plate Layouts for CellCiphr™
Cytotoxicity Profiling Multiplex Plates 1 and 2.
Figure 4. Data may be used to generate cluster map illustrating
cytotoxic responses of large numbers of compounds.
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Figure 1. Image pairs of DMSO control and compound treated
(24 hr) HepG2 cells.