Dye reagent; DNA Stain

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.

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]

---

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.

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).

[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 may be utilized for staining nuclei with blue fluorescence.

---

Cell Staining Example 2:
Hoechst 33342 may be utilized for staining nuclei with blue fluorescence to detect apoptosis.
1. Stain cells with Hoechst 33342 (10 min).
2. Observe with a fluorescence microscope (imaging).

---

Cell Staining Example 3:
Hoechst 33342 may be utilized for labelling chromatin with blue fluorescence.
1. Chromatin is counterstained briefly with Hoechst 33342 (5 µg/mL) before imaging.
2. Observe with a confocal microscope (imaging).

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).

---

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