1. Dye Reagents
  2. Hoechst 33258

Hoechst 33258 (Synonyms: bisBenzimide H 33258; H 33258)

Cat. No.: HY-15558
Handling Instructions

Hoechst 33258 is a fluorescent dye that emits blue fluorescence when bound to dsDNA.

For research use only. We do not sell to patients.

Hoechst 33258 Chemical Structure

Hoechst 33258 Chemical Structure

CAS No. : 23491-44-3

Size Price Stock
10 mM * 1 mL in DMSO USD 79 Ask For Quote & Lead Time
Solid + Solvent
10 mM * 1 mL
ready for reconstitution
USD 79 Ask For Quote & Lead Time
50 mg USD 72 Ask For Quote & Lead Time
100 mg USD 104 Ask For Quote & Lead Time

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Top Publications Citing Use of Products
  • Biological Activity

  • Protocol

  • Purity & Documentation

  • References

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Hoechst 33258 is a fluorescent dye that emits blue fluorescence when bound to dsDNA.

IC50 & Target

IC50: 51.31±4.56 μM (HeLa cell), 32.43±3.27 μM (HL60 cell), 15.42 ± 2.16 μM (U937 cell)[1]

In Vitro

Hoechst 33258, a fluorescent compound with a head-to-tail bis-benzimidazole structure, is initially found to be cytotoxic against L1210 murine leukemia. Hoechst 33258 is evaluated for their cytotoxicity against human tumor cell lines, which are cervix carcinoma cell line (HeLa), Human promyelocytic leukemia cell (HL60) and U937 cell Line. The IC50 determined in the case of HeLa, HL60 and U937 is 51.31±4.56, 32.43±3.27 and 15.42±2.16 μM for Hoechst 33258, respectively[1]. The cytotoxic property of Hoechst 33258 is investigated on a panel of seven tumour cell lines of different histological origin and Madine-Darby canine kidney (MDCK) normal cells. All cell lines, except MCF-7, exposed to Hoechst 33258 exhibit GI50 from 84×10-6 to 191.5×10-6 mol/dm3. Under the same experimental conditions, Hoechst 33258, used as a binder reference compound, stops the cell cycle in S phase and G0/G1[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Molecular Weight







Room temperature in continental US; may vary elsewhere.


-20°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

Solvent & Solubility
In Vitro: 

DMSO : 41.67 mg/mL (98.16 mM; ultrasonic and warming and heat to 60°C)

Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.3557 mL 11.7786 mL 23.5571 mL
5 mM 0.4711 mL 2.3557 mL 4.7114 mL
10 mM 0.2356 mL 1.1779 mL 2.3557 mL
*Please refer to the solubility information to select the appropriate solvent.
In Vivo:
  • 1.

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% saline

    Solubility: ≥ 2.08 mg/mL (4.90 mM); Clear solution

  • 2.

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in saline)

    Solubility: ≥ 2.08 mg/mL (4.90 mM); Clear solution

*All of the co-solvents are available by MCE.
Purity & Documentation

Purity: 99.93%

Cell Assay

Hoechst 33258 is prepared as stock solutions in highly pure water. Working solutions in a concentration range of 10-3-10-6 mol/dm3 are prepared prior to testing. Cytotoxic effects of Hoechst 33258 on tested cell lines are determined by the MTT assay. Cells are seeded in 96 micro well flat bottom plates at a concentration of 2×104 cells/mL and left overnight in the CO2 incubator allowing them to attach to the plate surface. Growing medium is replaced with compound supplemented or control medium and incubated for 72 h. Fresh medium with 5 mg/mL of MTT is added onto cells and incubated for 4 h at 37°C. Upon media removal, water insoluble MTT-formazan crystals formed inside the living cells are dissolved in DMSO and the absorbance at 570 nm proportional to the number of living cells is measured on an Elisa Microplate Reader. All experiments are performed at least three times in triplicates.The GI50 value, defined as the compound concentration (μM) leading to cellular growth inhibition by 50%, is calculated and used as a parameter to compare cytotoxicity among the compounds[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

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Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

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