HOECHST STAINS are part of a family of blue fluorescent dyes used to
* 1 Molecular characteristics * 2 Applications * 3 Toxicity and safety * 4 See also * 5 References * 6 External links
Excitation-emission spectra of Hoechst dyes.
Both dyes are excited by ultraviolet light at around 350 nm , and
both emit blue-cyan fluorescent light around an emission spectrum
maximum at 461 nm. Unbound dye has its maximum fluorescence emission
in the 510–540 nm range. Hoechst stains can be excited with a xenon-
or mercury-arc lamp or with an ultraviolet laser . There is a
Hoechst dyes are soluble in water and in organic solvents such as
dimethyl formamide or dimethyl sulfoxide . Concentrations can be
achieved of up to 10 mg/mL. Aqueous solutions are stable at 2–6 °C
for at least six months when protected from light. For long-term
storage the solutions are instead frozen at ≤-20 °C. Hoechst
33258 (magenta) bound to the minor groove of
The dyes bind to the minor groove of double-stranded
Transmission image of
A concentration of 0.1-12 µg/ml is commonly used to stain
Key differences between Hoechst dyes and
* Hoechst dyes are less toxic than DAPI, which ensures a higher viability of stained cells * The additional ethyl group of the Hoechst dyes renders them more cell-permeable. * There are nuclei staining dyes that allow for viability of cells after staining.
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
Hoechst dyes are commonly used to stain genomic
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.
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
* ^ Latt, SA; Stetten, G; Juergens, LA; Willard, HF; Scher, CD
(July 1975). "Recent developments in the detection of deoxyribonucleic
acid synthesis by 33258 Hoechst fluorescence.". Journal of
Histochemistry and Cytochemistry. 23 (7): 493–505. PMID 1095650 .
doi :10.1177/23.7.1095650 .
* ^ Latt, SA; Stetten, G (January 1976). "Spectral studies on 33258
Hoechst and related bisbenzimidazole dyes useful for fluorescent
detection of deoxyribonucleic acid synthesis". Journal of
Histochemistry and Cytochemistry. 24 (1): 24–33. PMID 943439 . doi
* ^ A B C "Hoechst Stains" (PDF). Invitrogren (Molecular Probes).
* ^ Portugal, J; Waring, MJ (Feb 28, 1988). "Assignment of DNA
binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide
(Hoechst 33258). A comparative footprinting study". Biochimica et
Biophysica Acta. 949 (2): 158–68. PMID 2449244 . doi
* ^ BD Bioscience (2009). Techniques for Immune Function Analysis
(PDF) (2 ed.). Becton, Dickinson and Company.
* ^ Kubbies, M; Rabinovitch, PS (January 1983). "Flow cytometric
analysis of factors which influence the BrdUrd-Hoechst quenching
effect in cultivated human fibroblasts and lymphocytes". Cytometry. 3
(4): 276–81. PMID 6185287 . doi :10.1002/cyto.990030408 .
* ^ Breusegem, SY; Clegg, RM; Loontiens, FG (Feb 1, 2002).
"Base-sequence specificity of Hoechst 33258 and