Hoechst stain
Hoechst stains are part of a family of blue
Molecular characteristics
Both dyes are excited by
Hoechst dyes are soluble in
The dyes bind to the
Applications
A concentration of 0.1–12 μg/ml is commonly used to stain DNA in bacteria or eukaryote cells. Cells are stained for 1-30 min at room temperature or 37 °C and then washed to remove unbound dye. A green fluorescence of unbound Hoechst dye may be observed on samples which are stained with too much dye or which are washed partially.[3] Hoechst dyes are often used as substitutes for another nucleic acid stain called DAPI.
Key differences between Hoechst dyes and DAPI are:
- Hoechst dyes are less toxic than DAPI, which ensures a higher viability of stained cells.[5]
- The additional ethyl group in certain Hoechst dyes (Hoechst 33342) renders them more cell-permeable.[6]
- There are nuclei staining dyes that allow for viability of cells after staining.[citation needed]
Hoechst 33342 and 33258 are quenched by bromodeoxyuridine (BrdU), which is commonly used to detect dividing cells. Hoechst 33342 exhibits a 10 fold greater cell-permeability than H 33258. Cells can integrate BrdU in newly synthesized DNA as a substitute for thymidine. When BrdU is integrated into DNA, it is supposed that the bromine deforms the minor groove so that Hoechst dyes cannot reach their optimal binding site. Binding of Hoechst dyes is even stronger to BrdU-substituted DNA; however, no fluorescence ensues. Hoechst dyes can be used with BrdU to monitor cell cycle progression.[7][8]
Hoechst dyes are commonly used to stain genomic DNA in the following applications:
- Flow cytometry to count or sort out cells. An example is the use of Hoechst dyes to analyse how many cells of a population are in which phase of the cell cycle[10]
- Detecting DNA in the presence of RNA in agarose gels[11]
- Automated DNA determination[12]
- Chromosome sorting[11]
Hoechst efflux is also used to study hematopoietic and embryonic stem cells. As these cells are able to effectively efflux the dye, they can be detected via flow cytometry in what is termed the side population. This is done by passing the fluorescence emitted from the excited hoechst through both red and blue filters, and plotting hoechst red and blue against each other.[citation needed]
Toxicity and safety
Because Hoechst stains bind to DNA, they interfere with DNA replication during cell division. Consequently, they are potentially mutagenic and carcinogenic, so care should be used in their handling and disposal. Hoechst stain is used to sort sperm in livestock and humans. Its safety has been debated.[13][14]
See also
References
- PMID 1095650.
- PMID 943439.
- ^ a b c "Hoechst Stains" (PDF). Invitrogren (Molecular Probes). Archived from the original (PDF) on 2009-04-19.
- PMID 2449244.
- ^ BD Bioscience (2009). Techniques for Immune Function Analysis (PDF) (2 ed.). Becton, Dickinson and Company.
- ISSN 2227-9040.
- PMID 6185287.
- PMID 11827475.
- ISBN 978-0-9829279-1-5.
- PMID 1692786.
- ^ PMID 2447564.
- PMID 2409841.
- PMID 7929716.
- PMID 15509707.