Hoechst stains are part of a family of blue fluorescent dyes used to
stain DNA. These Bis-benzimides were originally developed by
Hoechst AG, which numbered all their compounds so that the dye Hoechst
33342 is the 33342nd compound made by the company. There are three
related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst
34580. The dyes Hoechst 33258 and
Hoechst 33342 are the ones most
commonly used and they have similar excitation/emission spectra.
1 Molecular characteristics
3 Toxicity and safety
4 See also
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
Stokes shift between the excitation and emission spectra
that makes Hoechst dyes useful in experiments in which multiple
fluorophores are used. The fluorescence intensity of Hoechst dyes also
increases with the pH of the solvent.
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
Hoechst 33258 (magenta) bound to the minor groove of
DNA (green and
blue). From PDB: 264D.
The dyes bind to the minor groove of double-stranded
DNA with a
preference for sequences rich in adenine and thymine. Although the
dyes can bind to all nucleic acids, AT-rich double-stranded DNA
strands enhance fluorescence considerably. Hoechst dyes are
cell-permeable and can bind to
DNA in live or fixed cells. Thus, these
stains are often called supravital, meaning that live cells survive a
treatment with these compounds. Cells that express specific
ATP-binding cassette transporter
ATP-binding cassette transporter proteins can also actively transport
these stains out of their cytoplasm.
Transmission image of
HeLa cells, with overlay of Hoechst 33258
staining (blue). The leftmost cell is in the prometaphase stage of
mitosis; its chromosomes fluoresce brightly because they contain
highly compacted DNA.
Fluorescent image of cultivated neutrophils isolated from venous blood
of human with Alzheimer Disease. Sample was treated with Hoechst 33342
dye that is used to stain DNA. The picture shows the release of
a neutrophil as foggy area in the center of the view field indicating
the spontaneous activation of neutrophil extracellular traps formation
in AD patients that is not usually observed in healthy mates.
A concentration of 0.1-12 µg/ml is commonly used to stain
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.
Hoechst dyes are often used as substitutes for another nucleic acid
stain called DAPI.
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
There are nuclei staining dyes that allow for viability of cells after
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.
Hoechst dyes are commonly used to stain genomic
DNA in the following
Fluorescence microscopy and immunohistochemistry, often with other
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
DNA in the presence of RNA in agarose gels
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
Hoechst stain is used to sort sperm in livestock and
humans. Its safety has been debated.
Comparison of nucleic acid simulation software
^ 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. 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.
doi:10.1177/24.1.943439. PMID 943439.
^ a b c "Hoechst Stains" (PDF). Invitrogren (Molecular Probes).
Archived from the original (PDF) on 2009-04-19.
^ Portugal, J; Waring, MJ (Feb 28, 1988). "Assignment of
sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst
33258). A comparative footprinting study". Biochimica et Biophysica
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^ 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. doi:10.1002/cyto.990030408. PMID 6185287.
^ Breusegem, SY; Clegg, RM; Loontiens, FG (Feb 1, 2002).
"Base-sequence specificity of Hoechst 33258 and
DAPI binding to five
DNA sites with kinetic evidence for more than one high-affinity
Hoechst 33258-AATT complex". Journal of Molecular Biology. 315 (5):
1049–61. doi:10.1006/jmbi.2001.5301. PMID 11827475.
^ Iain Johnson, Michelle T.Z. Spence, ed. (2011). Molecular Probes
Handbook: A Guide to
Fluorescent Probes and Labeling Technologies (11
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^ Kubbies, M (1990). "Flow cytometric recognition of clastogen induced
chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst
fluorochrome binding". Cytometry. 11 (3): 386–94.
doi:10.1002/cyto.990110309. PMID 1692786.
^ a b Mocharla, R; Mocharla, H; Hodes, ME (Dec 23, 1987). "A novel,
sensitive fluorometric staining technique for the detection of
RNA preparations". Nucleic Acids Research. 15 (24): 10589.
doi:10.1093/nar/15.24.10589. PMC 339970 .
^ Sterzel, W; Bedford, P; Eisenbrand, G (June 1985). "Automated
DNA using the fluorochrome Hoechst 33258". Analytical
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^ Ashwood-Smith, M.J. (1994). "Safety of human sperm selection by flow
cytometry". Human Reproduction. Oxford University Press. 9 (5):
757–759. PMID 7929716.
^ Parrilla, I; Vázquez, J M; Cuello, C; Gil, MA; Roca, J; Di
Berardino, D; Martínez, EA (2004). "
Hoechst 33342 stain and u.v.
laser exposure do not induce genotoxic effects in flow-sorted boar
spermatozoa". Reproduction. 128 (5): 615–621.
doi:10.1530/rep.1.00288. PMID 15509707.
Wikimedia Commons has media related to Hoechst stains.
Spectral traces for fluorescent dyes
Manual for Hoechst stains
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