Hoechst (2(4-hydroxyphenyl)-5{5-(4-methylpiperazine-1-yl)-benzimidazol-2-yl}benzimidazole) is a type of bis-benzimidazole dye that binds to DNA. It is commonly used in the laboratory to analyze and quantify DNA in live or fixed cells. Hoechst dye comes in two structurally different forms: Hoechst 33258 and Hoechst 33342. The latter is a more lipophilic structure than the former and therefore can more easily pass through the cell's lipid membranes and reach the DNA. Hoechst 33342 can penetrate both live and fixed cells, while Hoechst 33258 can only penetrate cells that have been fixed and permeabilized by a detergent. Hoechst is not a natural compound but instead is a creation of a German laboratory.

Hoechst recognizes and forms noncovalent bonds with four or five base pairs on the DNA, preferably adenine-thymine (A-T) base pairs. It is commonly thought to be an intercalating agent, which is a compound that inserts itself in between base pairs and often distorts DNA. However this is incorrect, as Hoechst actually binds in the minor groove region and minimally distorts the DNA helix.

Besides binding DNA, Hoechst is also excited at UV wavelengths (351-364 nm) to emit a blue fluorescence at 465 nm. Therefore researchers can quantify and locate DNA in whole cells by first treating the cells with Hoechst and then exciting the dye with a UV laser.

Common uses for Hoechst dye include:

  • Determining the concentration of DNA in a solution. Hoechst dye increases its fluorescent intensity when it is bound to DNA and therefore can be used to quantify the amount of DNA in a solution. The solution, which must be free of contaminating RNA and proteins, is incubated with Hoechst and then analyzed in a fluorimeter. The amount of fluorescence produced by the Hoechst is used to determine the concentration of DNA.
  • Determining the location of the nucleus/chromosomes in fixed cells. Since Hoechst dye binds DNA it is a common marker used to locate the nucleus in interphase cells or the condensed chromosomes in mitotic cells. The fixed cells are incubated with the dye and then excited using a UV laser. The resulting blue fluorescence can be seen under a microscope to determine the location of the nucleus or chromosomes. Hoechst is also commonly used when performing immunofluorescence, a technique that uses fluorescent antibodies to locate a specific protein in fixed cells. Hoechst is added along with the antibody and both are analyzed simultaneously to determine if the protein is located in the nucleus or on condensed chromosomes.
  • Determining the cell cycle population of a sample of cells. As cells grow they move through the cell cycle from G1 phase to S phase to G2 phase and finally to mitosis where they divide. (See The mitotic cell cycle for a quick explanation of the phases). Researchers often want to determine what percentages of a group of cells are in G1, S, G2, or mitosis at a specific time. This percentage can be determined by using Hoechst. Cells that are in the G1 phase have one copy of their DNA, while cells that are in G2 phase and mitosis have two copies. Cells that are in S phase are actively copying their DNA and have a total amount between the G1 and G2/mitosis phases. The live or fixed cells are incubated with Hoechst and then analyzed by flow cytometry using a UV laser. Cells that are in G2/mitosis have twice the amount of fluorescence than cells in G1, while cells in S phase have an intermediate amount. The intensity of each cell is measured and used to determine the cell cycle population of the sample.

The pronounciation of Hoechst may vary a bit, but I've always heard it as "Hook-st"

Those who handle Hoechst should use extreme caution as it is a known carcinogen.


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