Direct measurement of nascent DNA synthesis is the most accurate method for determining cell proliferation in a population.  As a superior alternative to the traditional BrdU assays we offer our novel Click-iT® EdU Assays.  EdU (5-ethynyl-2´-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. Detection is accomplished by a copper-catalyzed covalent “click reaction” between the incorporated EdU and small azide derivatives of fluorescent dyes.  This provides a rapid, highly sensitive method for detecting DNA synthesis that is compatible with standard immunolabeling protocols.


Click-iT EdU Cell Proliferation Assay
Figure 1—Comparison of EdU direct detection and BrdU secondary detection. Rats were treated with estradiol three days prior to a 2 hr pulse of EdU or BrdU administered intraperiotoneally. Proliferating cells, labeled red, were detected either with a click reaction using Alexa Fluor® 594 azide supplied in the Click-iT® EdU Alexa Fluor® 594 Imaging Kit (left panel) or with anti-BrdU antibody followed by Alexa Fluor® 594 goat anti–mouse IgG secondary antibody (right panel). Nuclei are stained with the blue-fluorescent counterstain Hoechst 33342.
Product                               Cat. no.
Emission Color*
Ex/Em† Live‡ Fixed§ Fixable**
Click-iT® EdU Alexa Fluor® 488 High-Throughput Imaging (HCS) AssayA10027   350/461
495/519
 
 
 
Click-iT® EdU Alexa Fluor® 488 High-Throughput Imaging (HCS) AssayA10028 350/461
590/615
 
 
 
 
Click-iT™ EdU Alexa Fluor® 594 High-Throughput Imaging (HCS) AssayA10209 350/461
590/615
 
 
 
 
Click-iT® EdU Alexa Fluor® 594 High-Throughput Imaging (HCS) AssayC10082     350/461
590/615
 
 
Click-iT® EdU Alexa Fluor® 647 High-Throughput Imaging (HCS) AssayA10208    350/461
650/670
 
 
Click-iT® EdU Alexa Fluor® 647 High-Throughput Imaging (HCS) AssayC10081 350/461
650/670
 
 
* Gray represents fluorescence emission that is beyond the limit of human vision. † Ex/Em = Fluorescence excitation and emission maxima in nm. ‡ Stains live cells. § Stains fixed cells. ** Staining pattern is fixable. NA = Not applicable.
The HCS Mitotic Index Kit was developed to measure mitotic cells based on the levels of phospho- histone H3 the levels of which peak during mitosis.  This is an antibody based assay and as such can be used in combination with DNA profiling, general cytotoxicity, or antibody detection of choice targets. The kit provides a primary antibody against histone H3 (pS10) that is detected using a secondary antibody conjugated our Alexa Fluor® 488 dye. DAPI and HCS NuclearMask™ Deep Red stain are provided in the kit for DNA profiling and cell demarcation for image analysis.
HCS Mitotic Index Kit


Figure 2—Imaging of mitotic arrest in A549 cells with nocodazole using the HCS Mitotic Index Kit.
A549 cells were treated with 500 nM nocodazole for 24 hours at 37°C/5% CO2 and assayed using the HCS Mitotic Index Kit. Nuclear segmentation and DNA content measurements were done using DAPI or HCS NuclearMask™ Deep Red stain. At 500 nM, there was a strong increase in phospho-histone H3 staining indicative of mitotic cells. The images were quantitated using the Thermo Scientific Cellomics® ArrayScan® VTI platform.
Product                               Cat. no.
Emission Color*
Ex/Em† Live‡ Fixed§ Fixable**
HCS Mitotic Index KitH10293       365/450
495/519
638/686
 
 
* Gray represents fluorescence emission that is beyond the limit of human vision. † Ex/Em = Fluorescence excitation and emission maxima in nm. ‡ Stains live cells. § Stains fixed cells. ** Staining pattern is fixable. NA = Not applicable.
The HCS DNA Damage Kit was developed for measuring both genotoxicity and cytotoxicity in the same cell by high content screening and analysis. DNA damage is measured by specific antibody-based detection of phosphorylated H2AX (Ser139) in cell nuclei, which is detected by a highly cross-absorbed secondary antibody conjugated to the Molecular Probes® Alexa Fluor® 555 dye. For lethal cytotoxicity, the Image-iT® DEAD Green™ viability stain enables discrimination of dead cells by plasma membrane integrity. Hoechst 33342, which stains DNA in live and dead cells, is also provided in this kit as a segmentation tool for automated image analysis.
HCS DNA Damage Kit
Figure 3—Use of the HCS DNA Damage Kit to determine genotoxicity and cytotoxicity in A549 cells. Cells were treated with 30 µM or 120 µM valinomycin for 24 hours at 37°C/5% CO2 and toxicity was assayed using the HCS DNA Damage Kit. Imaging and analysis was performed using a 10X objective and the Compartmental Analysis Bioapplication with the Thermo Scientific Cellomics® ArrayScan® VTI platform. At 30 µM valinomycin, cells were positive for  pH2AX, but not for Image-iT® DEAD Green™ viability stain indicating DNA damage, but not a compromise in plasma  membrane integrity. At 120 µM valinomycin, cells showed genotoxic and cytotoxic effects as demonstrated by the positive pH2AX and Image-iT® DEAD Green™ viability stain fluorescence.
Product                               Cat. no.
Emission Color*
Ex/Em† Live‡ Fixed§ Fixable**
HCS DNA Damage KitH10292       350/461
488/515
550/565
 
 
† Ex/Em = Fluorescence excitation and emission maxima in nm. ‡ Stains live cells. § Stains fixed cells. ** Staining pattern is fixable. NA = Not applicable.