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]

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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.

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.

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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).

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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).

C32H37N7CL2.3[HCL]

699.97

697.179

159277-19-7

197365

Typically exists as solid at room temperature

841.2ºC at 760mmHg

1.59E-28mmHg at 25°C

8.726

5

5

11

44

784

0

Cl.Cl.Cl.ClCCN(C1C=CC(CCCC2=NC3C=CC(C4=NC5C=CC(N6CCN(C)CC6)=CC=5N4)=CC=3N2)=CC=1)CCCl

PLUDBJRFRHMTGA-UHFFFAOYSA-N

InChI=1S/C32H37Cl2N7.3ClH/c1-39-17-19-41(20-18-39)26-10-12-28-30(22-26)38-32(37-28)24-7-11-27-29(21-24)36-31(35-27)4-2-3-23-5-8-25(9-6-23)40(15-13-33)16-14-34;;;/h5-12,21-22H,2-4,13-20H2,1H3,(H,35,36)(H,37,38);3*1H

N,N-bis(2-chloroethyl)-4-[3-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]propyl]aniline;trihydrochloride

Hoechst 33342 analog trihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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