BrdU and Ki-67 Assay
Description:
BrdU and Ki-67 Assay
Product Overview:
Millipore’s HCS213 assay provides a complete immunofluorescence-based solution for identifying and quantifying BrdU incorporation and Ki-67 expression in cellular imaging applications. The assay may be used to study agents which modulate the cell cycle, proliferation index, cancer drug screening, and for in vitro toxicology studies. The reagents in the kit have been specifically optimized for HCS applications.
Background Information:
BrdU
Assessment of the proliferative state of a cell population has become an important parameter in drug discovery research, particularly in evaluating cancer therapeutics and in determining the health of a cell population during ADME/Tox studies. A traditional method for detection of cell proliferation is measurement of [3H] thymidine incorporation as cells enter S phase. This technology is slow, labor-intensive and has several limitations, including the handling and disposal of radioisotopes. A well established alternative to [3H] thymidine uptake uses bromodeoxyuridine (BrdU), a thymidine analog, to replace [3H] thymidine. When cells are pulsed with BrdU, it is incorporated into newly synthesized DNA strands of actively proliferating cells. The incorporation of BrdU into cellular DNA may then be detected using anti-BrdU antibodies, allowing assessment of the population of cells which are synthesizing DNA. This provides an indication of cell proliferation rate and a measurement of the number of cells in S phase during the pulse.
Ki-67
The Ki-67 antigen (Ki-67) is a classic marker of cellular proliferation and has found widespread application in diagnostic, research and drug discovery applications. The Ki-67 antigen was originally defined by the monoclonal antibody Ki-67, the name being derived from the city of origin (Kiel) and the number of the original clone in the 96-well plate. Ki-67 antigen is preferentially expressed during late G1, S, G2 and M phase of the cell cycle, while resting, non-cycling cells (G0 phase) lack Ki-67 expression. Thus, Ki-67 is commonly used as a proliferation marker.
Assessment of the proliferative state of a cell population has become an important parameter in drug discovery research, particularly in evaluating cancer therapeutics and in determining the health of a cell population during ADME/Tox studies. A traditional method for detection of cell proliferation is measurement of [3H] thymidine incorporation as cells enter S phase. This technology is slow, labor-intensive and has several limitations, including the handling and disposal of radioisotopes. A well established alternative to [3H] thymidine uptake uses bromodeoxyuridine (BrdU), a thymidine analog, to replace [3H] thymidine. When cells are pulsed with BrdU, it is incorporated into newly synthesized DNA strands of actively proliferating cells. The incorporation of BrdU into cellular DNA may then be detected using anti-BrdU antibodies, allowing assessment of the population of cells which are synthesizing DNA. This provides an indication of cell proliferation rate and a measurement of the number of cells in S phase during the pulse.
Ki-67
The Ki-67 antigen (Ki-67) is a classic marker of cellular proliferation and has found widespread application in diagnostic, research and drug discovery applications. The Ki-67 antigen was originally defined by the monoclonal antibody Ki-67, the name being derived from the city of origin (Kiel) and the number of the original clone in the 96-well plate. Ki-67 antigen is preferentially expressed during late G1, S, G2 and M phase of the cell cycle, while resting, non-cycling cells (G0 phase) lack Ki-67 expression. Thus, Ki-67 is commonly used as a proliferation marker.
Key Applications:
in vitro Toxicology Assays
Application Notes:
Cell Cycle
Species Reactivity:
Human
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.
Therapeutic Areas:
- Cardiovascular Disease
- Metabolic Disorders
Antigen:
BrdU and Ki-67 H3
Host Cells:
HeLa cell line
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., HeLa (human cervical adenocarcinoma, ATCC #CCL-2) or A549 (human lung carcinoma, ATCC #CCL-185).
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. 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., HeLa (human cervical adenocarcinoma, ATCC #CCL-2) or A549 (human lung carcinoma, ATCC #CCL-185).
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. 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.
Components:
- 1. Mouse Anti-BrdU HCS Primary Antibody, 5X
- 2. HCS Secondary Antibody (donkey anti-mouse IgG, FITC conjugate), 200X
- 3. Rabbit Anti-Ki-67 HCS Primary Antibody, 100X
- 4. HCS Secondary Antibody (donkey anti-rabbit IgG, Cy3 conjugate), 200X
- 5. Hoechst HCS Nuclear Stain, 200X
- 6. BrdU, 250X
- 7. HCS Fixation Solution with Phenol Red, 2X
- 8. HCS Immunofluorescence Buffer, 1X
- 9. HCS Wash Buffer, 1X
- 10. Aphidicolin (G1/S Arrest Control Compound), 250X
- 11. Etoposide (S/G2 Arrest Control Compound), 250X
- 12. Nocodazole (G2/M Arrest Control Compound), 250X
- 13. Paclitaxel (G2/M Arrest Control Compound), 250X
- 14. DMSO for Compound Serial Dilution
- 15. Compound Dilution Buffer
- 16. Plate Sealers
Kit Type:
High-Content Screening
Configuration:
Sufficient reagents for 5 X 96 well plates
Detection Methods:
Fluorescent
Kit or Assay Type:
High-Content Screening
Storage Conditions:
When stored under the conditions indicated on the labels, the kit components are stable up to the expiration date. HCS Fixation Solution, HCS Immunofluorescence Buffer, HCS Wash Buffer, DMSO, Compound Dilution Buffer, and Plate Sealers should be stored at 2-8ºC. HCS Primary Antibodies, HCS Secondary Antibodies, Hoechst HCS Nuclear Stain, BrdU, Aphidicolin, Etoposide, Nocodazole and Paclitaxel should be stored at -20ºC, avoiding repeated freeze/thaw cycles. (Note: If kit is expected to be used for multiple experiments, rather than a single use, thaw antibodies, nuclear stain and control compounds and dispense into appropriately sized aliquots. Store aliquots at -20ºC.)
Discard any remaining reagents after the expiration date.
Discard any remaining reagents after the expiration date.