Hoechst stain

Chemical structure of Hoechst dyes.

Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA.[1][2] 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.

Molecular characteristics

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 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 considerable 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.[3]

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.[3]

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.[4] Hoechst dyes are cell-permeable and can bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. Cells that express specific ATP-binding cassette transporter proteins can also actively transport these stains out of their cytoplasm.

Applications

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.

A concentration of 0.1-12 µg/ml is commonly used to stain DNA in bacteria or eukaryotic 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 were stained with too much dye or which were not washed thoroughly.[3] Hoechst dyes are often used as substitutes for another nucleic acid stain called DAPI.

Key differences between Hoechst dyes and DAPI are:

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 together with BrdU to monitor cell cycle progression.[6][7]

Hoechst dyes are commonly used to stain genomic DNA in the following applications:

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 known as 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 taken in their handling and disposal. Hoechst stain is used to sort sperm in livestock and humans. Its safety has been debated.[12][13]

See also

References

  1. ↑ 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.". The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 23 (7): 493–505. doi:10.1177/23.7.1095650. PMID 1095650.
  2. ↑ Latt, SA; Stetten, G (January 1976). "Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis". The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 24 (1): 24–33. doi:10.1177/24.1.943439. PMID 943439.
  3. 1 2 3 "Hoechst Stains" (PDF). Invitrogren (Molecular Probes).
  4. ↑ 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. doi:10.1016/0167-4781(88)90079-6. PMID 2449244.
  5. ↑ BD Bioscience (2009). Techniques for Immune Function Analysis (PDF) (2 ed.). Becton, Dickinson and Company.
  6. ↑ 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.
  7. ↑ Breusegem, SY; Clegg, RM; Loontiens, FG (Feb 1, 2002). "Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 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.
  8. ↑ Iain Johnson, Michelle T.Z. Spence, ed. (2011). Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies (11 ed.). Invitrogen. ISBN 0-9829279-1-6.
  9. ↑ 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.
  10. 1 2 Mocharla, R; Mocharla, H; Hodes, ME (Dec 23, 1987). "A novel, sensitive fluorometric staining technique for the detection of DNA in RNA preparations". Nucleic Acids Research 15 (24): 10589. doi:10.1093/nar/15.24.10589. PMC 339970. PMID 2447564.
  11. ↑ Sterzel, W; Bedford, P; Eisenbrand, G (June 1985). "Automated determination of DNA using the fluorochrome Hoechst 33258". Analytical Biochemistry 147 (2): 462–7. doi:10.1016/0003-2697(85)90299-4. PMID 2409841.
  12. ↑ Ashwood-Smith, M.J. (1994). "Safety of human sperm selection by flow cytometry". Human Reproduction (Oxford University Press) 9 (5): 757–759. PMID 7929716.
  13. ↑ 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.

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