Dye reagent; DNA Stain
Hoechst 33342 targets DNA, specifically binding to the minor groove of the double helix. It shows a preference for A/T-rich regions of DNA. Upon binding, its fluorescence is significantly enhanced, allowing for the visualization of DNA in live and fixed cells. It is a live-cell nuclear marker dye.

1. Preparation of Hoechst working solution
1.1: Preparation of Hoechst stock solution. Prepare 1 mg/mL Hoechst stock solution using DMSO.
*Note: After aliquot, Hoechst stock solution should be stored in the dark (protect from light) at -4°C or -20°C.
1.2: Preparing working solution: Dilute the stock solution with PBS or a serum-free cell culture medium to a 10 μg/mL of Hoechst working solution.
*Note: Before use, please make sure that the Hoechst working solution concentration is appropriate for your experiments, and use freshly prepared working solution for optimal results.
2. Staining (Suspended cells)
2.1: Centrifuge cells, add PBS, then wash twice for five minutes each time, or until the cell density reaches 1×106/mL.
2.2: Add 1 mL of Hoechst working solution and let it settle for 3–10 minutes.
2.3: Centrifuge for 3–4 minutes at 400 g, then discard supernatants.
2.4: Wash the cells twice with PBS, five minutes each time.
2.5: Re-suspend the cells in 1 mL of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe.
3. Staining (Adherent cell)
3.1: Grow adherent cells on sterile coverslips.
3.2: Remove the cover glass from the culture medium and remove excess culture medium.
3.3: Add 100 μ L of dye working solution, gently shake to completely cover the cells, and incubate for 3-10 minutes.
3.4: Remove the dye working solution, wash 2-3 times with culture medium for 5 minutes each time, and observe using a fluorescence microscope or flow cytometer. Note 1. Please adjust the concentration of Hoechst working solution according to the actual situation and prepare it for use.
2. This product is limited to the scientific research use of professional researchers and cannot be used for clinical diagnosis, treatment, food or medicine.
For your safety and health, please wear laboratory clothes and disposable gloves when operating.
In vitro, Hoechst 33342 is characterized by its DNA binding and fluorescent properties. When exposed to UV light, it emits bright fluorescence at two wavelengths: Hoechst Blue (405–450 nm) and Hoechst Red (630–650 nm). Its fluorescence is used to determine cell cycle status, conduct apoptosis assays, and act as a nuclear counterstain in microscopy.

In vivo, Hoechst 33342 is not a therapeutic agent. Its use is strictly in research settings, for applications such as labeling cells for tracking in live organisms or for staining tissues in histological studies. It is used to visualize cellular DNA in fixed or live cells for various research applications.

Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases. This protocol describes the use of Hoechst 33342 to label nuclear DNA of cells grown in culture. Hoechst 33342 can also be used to stain fixed cells by substituting Hoechst 33342 for DAPI.[1]
Hoechst 33342 can also be used to stain fixed cells by substituting Hoechst 33342 for DAPI in the protocol described in Labeling Nuclear DNA Using DAPI (Chazotte 2011a). Autofluorescence from endogenous cellular molecules such as the reduced forms of nicotinamide adenine dinucleotide or flavin adenine dinucleotide can interfere with imaging by reducing the signal-to-noise ratio. This occurs when the excitation and/or emission wavelengths of the probe and the autofluorescing molecules are similar, e.g., frequently with excitation wavelengths <500 nm, and particularly at ultraviolet wavelengths. Autofluorescence can be reduced by careful selection of the excitation and the emission wavelengths used, by treating fixed cells with reducing agents (e.g., a 1% solution of sodium borohydride [NaBH4] for 20 min), and by comparing the experimental images with unlabeled control slides. Avoid fixation with glutaraldehyde, because it can increase interference from cellular autofluorescence, most frequently at wavelengths <500 nm.[1]
This protocol assumes that the cells of interest were grown on glass microscope coverslips immersed in small Petri dishes containing culture medium. Generally, labeling conditions vary by cell type, and it might be necessary to alter the protocol for a particular use. To mount cells labeled using the technique described here, see Mounting Live Cells onto Microscope Slides (Chazotte 2011b).[3]

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A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells. One advantage of Hoechst 33342 is that it is membrane permeant and, thus, can stain live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases. This protocol describes the use of Hoechst 33342 to label nuclear DNA of cells grown in culture.
The in vitro binding assay for Hoechst 33342 is not a receptor assay but a DNA binding assay. The dye is incubated with purified DNA (e.g., calf thymus DNA) or with cells. Its fluorescence intensity is measured using a fluorometer or flow cytometer. The binding affinity can be assessed by titrating the dye with DNA and measuring the increase in fluorescence, from which a dissociation constant (Kd) can be calculated.

Method[1]
Do not allow the cells to dry out at any time during the protocol.
1. Dilute the Hoechst stock solution 1:100 in H2O for use in labeling.
2. Aspirate the cell medium from cells grown on coverslips. Rinse the cells three times with PBS+.
3. Incubate the cells in the Hoechst labeling solution (from Step 1) for 10-30 min at room temperature.
4. Aspirate the labeling solution. Rinse the cells three times in PBS+.
5. Mount the coverslips as described in Mounting Live Cells onto Microscope Slides (Chazotte 2011b).
In vitro cell-based assays using Hoechst 33342 are extensive. For cell cycle analysis, cells are stained with the dye, and the DNA content is measured by flow cytometry. Cells in G1 phase have a 2N DNA content, S phase have between 2N and 4N, and G2/M phase have 4N. For apoptosis, cells with fragmented DNA (sub-G1 peak) are detected. For nuclear counterstaining, cells are fixed and stained with the dye for visualization under a fluorescence microscope.

In vivo animal experiments using Hoechst 33342 are not standard for therapeutic testing. It is used as a research tool for cell labeling and tracking. For example, cells can be labeled with the dye and injected into an animal to study their migration, homing, and engraftment. The labeled cells can then be detected ex vivo in tissues using fluorescence microscopy or flow cytometry.

Hoechst 33342 trihydrochloride is a solid compound that is soluble in water and DMSO. It is typically stored as a powder at -20°C, protected from light. Stock solutions are prepared in water or DMSO. It is stable under recommended storage conditions. Its molecular weight is 561.93 g/mol.

Toxicological data for Hoechst 33342 indicate that it is a DNA-binding agent and can be genotoxic. It is also a known inhibitor of topoisomerase I. Therefore, it should be handled with care, and exposure should be minimized. It is not intended for human therapeutic use, and its use is restricted to in vitro and ex vivo research applications.

[1]. Chazotte B. Labeling nuclear DNA with hoechst 33342. Cold Spring Harb Protoc. 2011 Jan 1;2011(1):pdb.prot5557.
[2]. Chazotte B (2011a) Labeling nuclear DNA using DAPI. Cold Spring Harb Protoc doi:10.1101/pdb.prot5556.
[3]. Chazotte B (2011b) Mounting live cells onto microscope slides. Cold Spring Harb Protoc doi:10.1101/pdb.prot5554.

Cell Staining Example 1: Hoechst 33342 can be used for blue fluorescent staining of cell nuclei. Cell Staining Example 2: Hoechst 33342 can be used for blue fluorescent staining of cell nuclei to detect apoptosis. 1. Stain cells with Hoechst 33342 (10 minutes). 2. Observe using a fluorescence microscope (imaging). Cell Staining Example 3: Hoechst 33342 can be used for blue fluorescent labeling of chromatin. 1. Before imaging, briefly counterstain the chromatin with Hoechst 33342 (5 µg/mL). 2. Observe using a confocal microscope (imaging).
Hoechst 33342 is a standard and essential reagent in cell biology. It is a preferred nuclear counterstain for fluorescence microscopy because of its bright blue fluorescence and compatibility with other fluorescent proteins and dyes. It is also widely used in high-content screening and for the isolation of side population cells via flow cytometry.

C₂₇H₃₁CL₃N₆O

561.93

560.162

C, 57.71; H, 5.56; Cl, 18.93; N, 14.96; O, 2.85

875756-97-1

Hoechst 33342;23491-52-3;Hoechst 33342 analog;178481-68-0;Hoechst 33342 analog 2;106050-84-4

16760503

Light yellow to green yellow solid powder

7.332

5

5

5

37

664

0

JABNPSKWVNCGMX-UHFFFAOYSA-N

InChI=1S/C27H28N6O.3ClH/c1-3-34-21-8-4-18(5-9-21)26-28-22-10-6-19(16-24(22)30-26)27-29-23-11-7-20(17-25(23)31-27)33-14-12-32(2)13-15-33;;;/h4-11,16-17H,3,12-15H2,1-2H3,(H,28,30)(H,29,31);3*1H

2'-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride

bisBenzimide; H 33342 trihydrochloride; HOE 33342 trihydrochloride; HOE 33342 trihydrochlorde; HO342; Hoechst-33342; Hoechst33342; Hoechst 33342; NSC 334072.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 46 mg/mL (~81.86 mM)
H2O : ≥ 5.6 mg/mL (~9.97 mM)

Solubility in Formulation 1: 3.33 mg/mL (5.93 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)