CellCiphr Cytotoxicity Assay Human HepG2 Cells Cellular Systems Biology (CSB) Kit

Description: CellCiphr Cytotoxicity Assay Human HepG2 Cells Cellular Systems Biology (CSB) Kit

Background Information:
Millipore and Cellumen have developed the CellCiphr Cytotoxicity Assay for human HepG2 cells, a multiplexed Cellular Systems Biology Profiling kit comprised of high-quality, validated, automation-compatible detection reagents and validated protocols for profiling multiple human hepatotoxicity endpoints. The CellCiphr Cytotoxicity Assay for human HepG2 cells provides a means to screen compounds for a broad range of potentially toxic effects early in the drug discovery process, and provide better information to drive drug development.

Key Applications: Immunocytochemistry

Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Sensitivity:

• Millipore and Cellumen's HCS100 CellCiphr Cytotoxicity Assay for human HepG2 cells is a Cellular Systems Biology Profiling kit for multiparametric analysis of drug-induced human cytotoxicity. Parameters were selected to monitor the effects of test compounds on many cellular systems responses known to be correlated with toxic challenge. Eleven cellular responses may be analyzed using this assay kit.
• 1. Cell Loss.
• 2. Cell Cycle Arrest.
• 3. DNA Degradation.
• 4. Nuclear Size.
• 5. Oxidative Stress.
• 6. Stress Pathway Activation.
• 7. DNA Damage response.
• 8. Mitochondrial Membrane Potential.
• 9. Mitochondrial Mass.
• 10. Mitotic Arrest.
• 11. Cytoskeletal Integrity.
• The assay has been designed to allow detection of all 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. The kit also includes buffers for Compound Dilution, Cell Fixation, Cell Permeabilization, Antibody Dilution and Washing, as well as disposable syringes and needles for HepG2 cell handling and plate sealers. All materials and protocols provided have been validated for multiplexed cytotoxicity analysis on both the GE IN Cell Analyzer 1000 and the Cellomics ArrayScan VTI.
• The assay has been designed to be performed in 6 tissue-culture treated 384-well microplates, with two plates required for each timepoint. See Figure 2 for multiplexed plate layout.
• <bold>Table 1.</bold> Antibody Selection and Cellular Response. Cytotox Profile: Multiplex Plate 1
• <LB/>table border="1"<RB/> <LB/>tr<RB/> <LB/>th colspan="3"<RB/><bold>CellCiphr Multiplex Plate 1<LB/>/th<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/><bold>Cell Parameter</bold><LB/>/td<RB/> <LB/>td<RB/><bold>Measurement</bold><LB/>/td<RB/> <LB/>td<RB/><bold>Detection</bold><LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Cell Loss<LB/>/td<RB/> <LB/>td<RB/>Cell number<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Cell Cycle Arrest<LB/>/td<RB/> <LB/>td<RB/>DNA Content<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>DNA Degradation<LB/>/td<RB/> <LB/>td<RB/>DNA Structure<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Nuclear Size<LB/>/td<RB/> <LB/>td<RB/>Nuclear Area<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Oxidative Stress<LB/>/td<RB/> <LB/>td<RB/>Phosphorylation<LB/>/td<RB/> <LB/>td<RB/>Phospho-Histone H2A.X<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Stress Pathway Activation<LB/>/td<RB/> <LB/>td<RB/>Phosphorylation<LB/>/td<RB/> <LB/>td<RB/>Phospho-c-jun<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>DNA Damage Response<LB/>/td<RB/> <LB/>td<RB/>Target Activation<LB/>/td<RB/> <LB/>td<RB/>p53<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>/table<RB/>
• <bold>Table 2. </bold> Antibody Selection and Cellular Response. Cytotox Profile: Multiplex Plate 2
• <LB/>table border="1"<RB/> <LB/>tr<RB/> <LB/>th colspan="3"<RB/><bold>CellCiphr Multiplex Plate 2<LB/>/th<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/><bold>Cell Parameter</bold><LB/>/td<RB/> <LB/>td<RB/><bold>Measurement</bold><LB/>/td<RB/> <LB/>td<RB/><bold>Detection</bold><LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Cell Loss<LB/>/td<RB/> <LB/>td<RB/>Cell number<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Cell Cycle Arrest<LB/>/td<RB/> <LB/>td<RB/>DNA content<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>DNA Degradation<LB/>/td<RB/> <LB/>td<RB/>DNA Structure<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Nuclear size<LB/>/td<RB/> <LB/>td<RB/>Nuclear area<LB/>/td<RB/> <LB/>td<RB/>Nuclear Stain<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Mitochondrial function I<LB/>/td<RB/> <LB/>td<RB/>Mitochondrial membrane potential<LB/>/td<RB/> <LB/>td<RB/>MitoDye<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Mitochondrial function II<LB/>/td<RB/> <LB/>td<RB/>Mitochondrial Mass<LB/>/td<RB/> <LB/>td<RB/>MitoDye<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Mitotic Arrest<LB/>/td<RB/> <LB/>td<RB/>Phosphorylation<LB/>/td<RB/> <LB/>td<RB/>Phospho-Histone H3<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>tr<RB/> <LB/>td<RB/>Cytoskeletal Integrity<LB/>/td<RB/> <LB/>td<RB/>Microtubule Stability<LB/>/td<RB/> <LB/>td<RB/>α-Tubulin<LB/>/td<RB/> <LB/>/tr<RB/> <LB/>/table<RB/>

Packaging: Each assay provides enough reagents for 16 compounds measurements at three time points

Materials Required but Not Delivered:
1. HepG2 cells. Available from ATCC, catalog number HB-8065.


2. Tissue culture equipment/supplies for culturing HepG2 cells (e.g., 37°C incubator, sterile cell culture hood, growth media, Trypsin-EDTA solution, tissue culture flasks/plates, sterile centrifuge tubes, pipettes, etc).


3. 384-well microplates suitable for High Content Imaging of HepG2 cells.


4. Dimethyl sulfoxide (DMSO). HPLC grade is recommended.


5. High Content Imaging hardware and software, e.g., GE IN Cell Analyzer 1000, Cellomics ArrayScan VTI, Evotec Opera. Imaging system must be equipped with beam-splitters and filters capable of reading emission spectra in the blue, green, red and far red ranges. Example filter ranges:

Fluorescent Reagent Excitation Filter Range [peak/bandwidth (nm)] Emission Filter Range [peak/bandwidth (nm)]
Nuclear Stain 360/40 460/40 (or 535/50 if necessary)
FITC 475/20 535/50
Cy3/MitoDye 535/50 600/50
Cy5 620/60 700/75

Components:

• Antibody A, 200x. Anti-phospho-histone H2A.X
• Antibody B, 200x. Anti-Antibody A, FITC conjugate
• Antibody C, 200x. Anti-phospho-c-jun
• Antibody D, 200x. Anti-Antibody C, Cy3 conjugate
• Antibody E, 200x. Anti-p53
• Antibody F, 200x. Anti-Antibody E, Cy5 conjugate
• Antibody G, 200x. Anti-phospho-histone H3
• Antibody H, 200x. Anti-Antibody G, FITC conjugate
• Antibody I, 200x. Anti-α-tubulin
• Antibody J, 200x. Anti-Antibody I, Cy5 conjugate
• MitoDye solution, 10,000x
• Nuclear Stain Solution, 200x
• Compound Dilution Buffer
• Cell Fixation Buffer
• Cell Permeabilization Buffer
• Antibody Dilution Buffer
• Wash Buffer
• Control Toxin 1, Anisomycin, 2.5 mM in DMSO, 250x
• Control Toxin 2, Camptothecin, 2.5 mM in DMSO, 250x
• Control Toxin 3, CCCP, 25 mM in DMSO, 250x
• Control Toxin 4, Paclitaxel, 0.25 mM in DMSO, 250x
• 20 gauge needle with syringe, 5cc capacity
• Plate Sealers
• xCellCiphr Data Conversion Tool CD

Storage Conditions: 1 year at -20°C from date of shipment