HOE-S 785026 tri HCl is a marking dye from the Hoechst series. The Hoechst series are live cell nuclear labeling dyes. Hoechst binds nucleic acids via binding to the minor groove in the DNA double strand. Hoechst prefers to bind to A/T-rich DNA strands. At the same time, A/T-rich double-stranded DNA significantly enhances the fluorescence intensity. Hoechst can cross cell membranes and bind to living or fixed cells. The fluorescence intensity of Hoechst dye increases with increasing solution pH.

Physicochemical Properties

Molecular Formula	C25H27CL3N6O
Molecular Weight	533.880481958389
Exact Mass	532.131
CAS #	2320308-12-9
Related CAS #	HOE-S 785026;132869-83-1
PubChem CID	131704504
Appearance	Light green to green solid powder
Hydrogen Bond Donor Count	6
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	3
Heavy Atom Count	35
Complexity	642
Defined Atom Stereocenter Count	0
InChi Key	ZWKREDJJELOWKN-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H24N6O.3ClH/c1-30-9-11-31(12-10-30)18-6-8-21-23(15-18)29-25(27-21)17-5-7-20-22(14-17)28-24(26-20)16-3-2-4-19(32)13-16;;;/h2-8,13-15,32H,9-12H2,1H3,(H,26,28)(H,27,29);3*1H
Chemical Name	3-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]phenol;trihydrochloride
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

Hoechst working solution preparation 1.1 Hoechst stock solution preparation Make a 1 mg/mL Hoechst stock solution using DMSO. Note: After aliquots, Hoechst storage solution should be kept at -4°C or -20°C out of direct sunlight. 1.2 Making the working solution: Use either PBS-diluted storage solution or heated serum-free cell culture medium, bringing the final concentration of the Hoechst working solution to 10 μg/mL. Note: Before using, please make sure that the Hoechst working fluid concentration is appropriate for the current circumstances. 2. Suspended cell staining (2.1): Centrifuge cells, add PBS, then wash twice for five minutes each time. The density of cells is 1×106/mL. 2.2 After adding 1 mL of Hoechst working solution, incubate for 3–10 minutes at room temperature. 2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Adherent cell staining (3.1): Grow adherent cells on sterile coverslips. 3.2 Aspirate extra culture medium after removing the coverslip from the medium. 3.3 Add 100 μL of the dye working solution, give the cells a full shake to cover them, and then let them sit for three to ten minutes. 3.4 Use a flow cytometer or fluorescence microscope to observe after aspirating the dye working solution and washing it twice in culture media for a total of five minutes each time. Observations 1. Kindly modify the Hoechst working fluid concentration based on the current circumstances. 2. This product may not be used for clinical diagnosis or treatment, nor may it be included into food or medication. It is intended solely for professional use in scientific study. 3. Please wear disposable gloves and a lab coat for your health and safety.

Solubility Data

Solubility (In Vitro)

H2O: 47.3 mg/mL (88.60 mM)

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8731 mL	9.3654 mL	18.7308 mL
	5 mM	0.3746 mL	1.8731 mL	3.7462 mL
	10 mM	0.1873 mL	0.9365 mL	1.8731 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.