What is the Difference Between DAPI and Hoechst

The key difference between DAPI and Hoechst is that DAPI is a dye that is useful in staining DNA content in fixed cells, while Hoechst is a dye that is useful in staining DNA content in live cells.

Differential staining of the cell cycle is a very important method. It allows the staining of DNA and RNA, permitting the determination of DNA or RNA content in cells. This, in turn, allows one to discriminate different phases in the cell cycle, such as G(0), G(1), and cell differentiation. There are different stains for the differential staining of cells. DAPI and Hoechst are two types of stains used in differential staining of cells.

CONTENTS

1. Overview and Key Difference
2. What is DAPI 
3. What is Hoechst
4. Similarities – DAPI and Hoechst
5. DAPI vs Hoechst in Tabular Form
6. Summary – DAPI vs Hoechst

What is DAPI?

DAPI is a fluorescent stain. It binds very strongly to adenine and thymine-rich regions in the DNA. This stain is extensively used in fluorescence microscopy. This stain is also known as DAPPY or 4, 6-diamidino-2-phenylindole. As DAPI can pass through an intact cell membrane, it can be used to stain DNA content in cells. However, it has low cell permeability, so it is generally used to stain DNA content in fixed cells. DAPI is excited by ultraviolet light at around a wavelength of 358 nm, and it emits blue-cyan fluorescent light around an emission spectrum maximum of 461 nm.

DAPI was first synthesized in 1971 in the laboratory of Otto Dann, Erlangen, as a drug against trypanosomiasis disease. But it was unsuccessful as a drug. Further investigation revealed that it binds strongly to DNA and becomes more fluorescent when bound to DNA. This led to its use in identifying DNA content. The first recorded use of DAP1 as a fluorescent DNA stain was in the year 1975 in identifying mitochondrial DNA in ultracentrifugation.

Furthermore, DAPI is normally labeled as non-toxic in its MSDS (material safety data sheet). Anyhow, as it is a small DNA-binding compound, it is likely to have some carcinogenic effects. Therefore, care should be taken when handling and disposing of DAPI stains.

What is Hoechst?

Hoechst is a part of the family of blue fluorescent dyes. It has high cell permeability, so it can be used to detect the DNA content of live cells. Hoechst is a bisbenzimide, which is an organic compound. There are normally three related Hoechst stains used in DNA staining: Hoechst 33258, Hoechst 33342, and Hoechst 34580. However, the dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used in laboratories. Moreover, Hoechst 33258 and Hoechst 33342 have similar excitation–emission spectra. Both these dyes are excited by ultraviolet light at around a wavelength of 350 nm. They emit blue-cyan fluorescent light around an emission spectrum maximum of 461 nm.

Hoechst has different applications: fluorescent microscopy and immunohistochemistry, flow cytometry to count or sort out cells, differential staining of the cell cycle, detecting DNA in the presence of RNA in agarose gels, automated DNA determination, and chromosome sorting. Furthermore, they can interfere with DNA replication during cell division, so they are potentially mutagenic and carcinogenic. Therefore, care should be taken when handling and disposing of Hoechst stain.

What are the Similarities Between DAPI and Hoechst?

• DAPI and Hoechst are two types of stains used for the differential staining of cells.
• They are blue fluorescent dyes.
• Both stains prefer binding with the A/T-rich regions in DNA.
• They are minor grove-binding stains.
• Both stains are visible using the same equipment filter settings.
• They can be carcinogenic.

What is the Difference Between DAPI and Hoechst?

DAPI is a dye that is typically used to stain DNA content in fixed cells, while Hoechst is a dye that is typically used to stain DNA content in live cells. Thus, this is the key difference between DAPI and Hoechst. Furthermore, DAPI has low cell permeability, while Hoechst has high cell permeability.

The below infographic presents the differences between DAPI and Hoechst in tabular form for side-by-side comparison.

Summary – DAPI vs Hoechst

DNA staining is a very important aspect in determining the DNA content of a cell. DAPI and Hoechst are two blue fluorescent dyes used for the differential staining of cells. Both these stains are used in fluorescent microscopy, and they are minor grove-binding stains in DNA. However, DAPI is typically used for staining DNA content in fixed cells due to its low membrane permeability, while Hoechst is typically used for staining DNA content in live cells due to its high membrane permeability. So, this summarizes the difference between DAPI and Hoechst.

Reference:

1. “Dapi.” An Overview | ScienceDirect Topics.
2. Scharenberg, Christian W., et al. “ABCG2 Transporter Is an Efficient Hoechst 33342 Efflux Pump and Is Preferentially Expressed by Immature Human Hematopoietic Progenitors.” American Society of Hematology.

Image Courtesy:

1. “38F3-ChkNFH-DAPI-Shsy5y” By GerryShaw – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Structure of Hoechst dyes” By Crenim – Own work (CC0) via Commons Wikimedia