Hoechst 33342 *Ultrapure Grade* *CAS 23491-52-3*

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<strong>Workflow for the “three-in-one” cell death screening assay. </strong>HUVECs growing in the 96-well plate for 48 hours are exposed to NPs for 24 hours. Three types of cell death are evaluated simultaneously. A) Cell necrosis is measured spectrophotometrically after mixing an aliquot of cell supernatant with LDH substrate. B) Cell viability is assessed by adding WST-8 substrate to the cells. After three hours of incubation, aliquots of the reaction mixture are transferred into the new plate and measured spectrophotometrically. C) Cell apoptosis is detected after incubating the cells with Hoechst 33342 and fixing them with paraformaldehyde. Images captured under the inverted fluorescence microscope are computationally processed with the specially designed ImageJ macro. Source: <strong>An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells</strong> by Marcela Filipova et al., <em>PLOS</em>, Oct. 2018.
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Ex/Em (nm)350/451
CAS #23491-52-3
Storage Freeze (<-15 °C)
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Category DNA and RNA,
Biomolecule Detection,
Cell Imaging
The Hoechst stains are a family of fluorescent stains for labeling DNA in fluorescence microscopy. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. Two of these closely related bis-benzimides are commonly used: Hoechst 33258 and Hoechst 33342. Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescence light around an emission maximum at 461 nm. The Hoechst stains may be used on live or fixed cells, and are often used as a substitute for another nucleic acid stain, DAPI. The key difference between them is that the additional ethyl group of Hoechst 33342 renders it more lipophilic, and thus more able to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant. These dyes can also be used to detect the contents of a sample DNA by plotting a standard emission-to-content curve.

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of Hoechst 33342 *Ultrapure Grade* *CAS 23491-52-3* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
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This protocol only provides a guideline, and should be modified according to your specific needs.

The following procedure can be adapted for most cell types. Growth medium, cell density, the presence of other cell types and other factors may influence staining. Residual detergent on glassware may also affect real or apparent staining of many organisms, causing brightly stained material to appear in solutions with or without cells present.

Pellet cells by centrifugation and resuspend the cells in buffered salt solutions or media, with optimal dye binding at pH 7.4. Adherent cells in culture may be stained in situ on cover slips or in the cell culture wells. Add Hoechst stain using the concentrations between 0.5 and 5 µM and incubate it for 15 to 60 minutes as a guide. In initial experiments, it may be best to try several dye concentrations over the entire suggested range to determine the concentration that yields optimal staining.

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Alternative forms
Product nameCurrent ProductRelated Product
Hoechst 33342 *20 mM solution in water*
Ultrapure Grade CAS 23491 52 3
20 mM solution in water


An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells
Authors: Marcela Filipova, Oumsalama K Elhelu, Silvia H De Paoli, Zuzana Fremuntova, Tibor Mosko, Dusan Cmarko, Jan Simak, Karel Holada
Journal: PloS one (2018): e0206557

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