Informazioni per l’ordine
NeuroTox-3: High Content Analysis Kit for Co-Culture of Neurons and Astrocytes
Description:
NeuroTox-3: High Content Analysis Kit for Co-Culture of Neurons and Astrocytes
Product Overview:
Millipore’s HCS222 High Content Analysis kit for co-culture of neurons and astrocytes is comprised of high-quality, validated, target-specific detection reagents for profiling βIII-tubulin and glial fibrillary acidic protein (GFAP), enabling analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models. The highly-validated reagents provided with this kit allow the user to standardize assays, minimize assay-to-assay variability, and to reproducibly generate images with a high signal-to-background ratio.
The assay is designed to enable visualization and quantitative detection of βIII-tubulin and GFAP, allowing the characterization of compounds that may induce or inhibit neuronal and astrocytic development and injury. The nuclear dye (Hoechst 33342) may be used for measurements of cell number, DNA content and nuclear size. Additionally, the assay can be multiplexed with other probes, e.g., for signaling pathway studies, drug efficacy trials or in vitro toxicology applications.
The assay is designed to enable visualization and quantitative detection of βIII-tubulin and GFAP, allowing the characterization of compounds that may induce or inhibit neuronal and astrocytic development and injury. The nuclear dye (Hoechst 33342) may be used for measurements of cell number, DNA content and nuclear size. Additionally, the assay can be multiplexed with other probes, e.g., for signaling pathway studies, drug efficacy trials or in vitro toxicology applications.
Background Information:
Neuronal quantitation in in vitro models of neural development and neurotoxicity.
The use of HCS for measurement of neurite outgrowth and neuronal biology has become a valuable tool for neuroscience research, drug discovery and neurotoxicity screening [9-17]. A large number of neural cell types have been successfully utilized for HCS. These include immortalized/stable cell lines such as PC12, SHSY5Y and N2A; primary hippocampal, cortical and dorsal root ganglia (DRG) neurons; cerebellar granular cells and neuronal stem cells. By labeling neuronal cells with an antibody recognizing a neuronal-specific marker such as βIII-tubulin, HCS provides the opportunity to perform high-throughput, non-subjective, quantitative neuronal assays, and enables morphological screening of multiple parameters in individual cells. Parameters which may be readily detected using HCS platforms include neuronal number, neurite count, neurite length, neurite area and branch point count. Accordingly, HCS has been demonstrated to be an effective method to screen for inducers of neurite outgrowth and neuronal regeneration. Compared to traditional approaches which are labor-intensive and subjective, HCS-based neurite outgrowth assays offer the opportunity for greatly improved productivity in neuroscience research and drug discovery.
Glial fibrillary acidic protein (GFAP) as a biomarker of neurotoxicity amenable to High Content Screening.
GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the central nervous system (CNS). Astrocytes have an integral role in the maintenance of CNS homeostasis, and are also associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. Astrocytic activation (gliosis) leads to the upregulation of GFAP commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system and can be detected morphologically or quantified by measurements of GFAP.
The use of HCS for measurement of neurite outgrowth and neuronal biology has become a valuable tool for neuroscience research, drug discovery and neurotoxicity screening [9-17]. A large number of neural cell types have been successfully utilized for HCS. These include immortalized/stable cell lines such as PC12, SHSY5Y and N2A; primary hippocampal, cortical and dorsal root ganglia (DRG) neurons; cerebellar granular cells and neuronal stem cells. By labeling neuronal cells with an antibody recognizing a neuronal-specific marker such as βIII-tubulin, HCS provides the opportunity to perform high-throughput, non-subjective, quantitative neuronal assays, and enables morphological screening of multiple parameters in individual cells. Parameters which may be readily detected using HCS platforms include neuronal number, neurite count, neurite length, neurite area and branch point count. Accordingly, HCS has been demonstrated to be an effective method to screen for inducers of neurite outgrowth and neuronal regeneration. Compared to traditional approaches which are labor-intensive and subjective, HCS-based neurite outgrowth assays offer the opportunity for greatly improved productivity in neuroscience research and drug discovery.
Glial fibrillary acidic protein (GFAP) as a biomarker of neurotoxicity amenable to High Content Screening.
GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the central nervous system (CNS). Astrocytes have an integral role in the maintenance of CNS homeostasis, and are also associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. Astrocytic activation (gliosis) leads to the upregulation of GFAP commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system and can be detected morphologically or quantified by measurements of GFAP.
Key Applications:
- High Content Analysis
- Immunofluorescence
- in vitro Toxicology Assays
- Neuroscience
- Neurotoxicity
Application Notes:
Co-culture of neurons and astrocytes, Neuronal differentiation
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.
Kit or Assay Type:
- High-Content Screening
- High-Content Analysis
Configuration:
Sufficient reagents for 5 X 96 well plates
Components:
- 1. Rabbit Anti-βIII-Tubulin HCS Primary Antibody, 100X
- 2. HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X
- 3. Mouse Anti-GFAP HCS Primary Antibody, 100X
- 4. HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X
- 5. Hoechst HCS Nuclear Stain, 200X
- 6. HCS Fixation Solution with Phenol Red, 2X
- 7. HCS Immunofluorescence Buffer, 1X
- 8. HCS Wash Buffer, 1X
- 9. Acrylamide (Control Compound), 10X
- 10. Hydrogen Peroxide (Control Compound), 10X
- 11. K-252a (Control Compound), 250X
- 12. DMSO for Compound Serial Dilution
- 13. Compound Dilution Buffer
- 14. Plate Sealers
Storage Conditions:
Store kit components under the conditions indicated on the labels. Discard any remaining reagents after 6 months.
Detection Methods:
Fluorescent
Antibody Category:
Neuroscience
Materials Required but Not Delivered:
1. Sterile, tissue culture-treated black/clear bottom microplates suitable for High-Content Imaging.
2. Cell-type for assay, e.g., PC12 (rat pheochromocytoma, ATCC #CRL-1721), SHSY5Y (human neuroblastoma, ATCC #CRL-2266), primary human or rodent astrocytes/neurons; astrocytoma or glioma cell lines.
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. Sterile distilled or deionized water (dH2O) for Control Compound dilution, as appropriate.
5. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. Imaging system must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.
2. Cell-type for assay, e.g., PC12 (rat pheochromocytoma, ATCC #CRL-1721), SHSY5Y (human neuroblastoma, ATCC #CRL-2266), primary human or rodent astrocytes/neurons; astrocytoma or glioma cell lines.
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. Sterile distilled or deionized water (dH2O) for Control Compound dilution, as appropriate.
5. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. Imaging system must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.