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| ln Vitro |
Hoechst working solution preparation 1.1 Hoechst stock solution preparation Make a 1 mg/mL Hoechst stock solution using DMSO. Note: After filling, Hoechst storage solution should be kept in the dark at -4°C or -20°C. 1.2 Replacing the working solution: Before using the storage solution, add PBS or a high-quality serum-free cell culture medium. Ten μg/mL of Hoechst working solution is the final concentration. 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. Add 1 mL of Hoechst working solution and let it settle for 3–10 minutes, or until the cell density reaches 1×106/mL 2.2. 2.3 Centrifuge for 3–4 minutes at 400 g, then discard. 2.4 Wash the cells twice with PBS, giving them five minutes each time. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free water, and use a flow cytometer or fluorescence microscope to observe. 3. Adhesion-based cell staining 3.1 Grow adherent cells on sterile coverslips. 3.2 Aspirate extra cells and remove the coverslip from the culture medium. Step 3: Aspirate the dye working solution, wash the cells 2-3 times with culture media for 5 minutes each time, and use a fluorescence microscope or flow cytometer to monitor. Step 3.3 Add 100 μL of dye working solution and shake gently to completely cover the cells. Observations 1. Kindly modify the Hoechst working fluid concentration based on the current circumstances. Ten minutes. 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. When working, please wear gloves and a lab coat for your own health and safety.
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| References |
[1]. Latt SA, Stetten G, Juergens LA, Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 23 (7): 493-505.
[2]. a b c "Hoechst Stains". Invitrogren (Molecular Probes). [3]. Portugal J, Waring MJ. Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study. Biochimica et Biophysica Acta 949 (2): 158-68. |
| Molecular Formula |
C29H26N6
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|---|---|
| Molecular Weight |
458.5569
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| CAS # |
23491-55-6
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| Related CAS # |
Hoechst 33258;23491-44-3;Hoechst 33258 analog;258843-62-8
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| Appearance |
Typically exists as solids (or liquids in special cases) at room temperature
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| SMILES |
N1(C2C([H])=C([H])C3=C(C=2[H])N([H])C(C2C([H])=C([H])C4=C(C=2[H])N([H])C(C2C([H])=C([H])C5=C([H])C([H])=C([H])C([H])=C5C=2[H])=N4)=N3)C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H]
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~33.33 mg/mL (~72.68 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.45 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1807 mL | 10.9037 mL | 21.8074 mL | |
| 5 mM | 0.4361 mL | 2.1807 mL | 4.3615 mL | |
| 10 mM | 0.2181 mL | 1.0904 mL | 2.1807 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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