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> Hoechst 33258 [23491-44-3]

Hoechst 33258 [23491-44-3]

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Cat# HY-15558-100mg

Size : 100mg

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Hoechst 33258 is a marker dye in Hoechst series. Hoechst is A live nuclear marker dye. Hoechst binds to the grooves in the DNA double strand, which tends to be A/ T-rich DNA strand. Although it binds to all nucleic acids, the A/ T-rich double strand DNA significantly enhances fluorescence intensity Therefore,Hoechst dye can be used for living cell labeling. The fluorescence intensity of Hoechst dye increases with the increase of pH of solution.

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

Hoechst 33258 Chemical Structure

Hoechst 33258 Chemical Structure

CAS No. : 23491-44-3

This product is a controlled substance and not for sale in your territory.

Based on 14 publication(s) in Google Scholar

Other Forms of Hoechst 33258:

  • Hoechst 33258 analog In-stock
  • Hoechst 33258 analog 2 In-stock
  • Hoechst 33258 analog 3 In-stock
  • Hoechst 33258 analog 5 In-stock
  • Hoechst 33258 analog 6 In-stock
  • Hoechst 33258 trihydrochloride In-stock
Description

Hoechst 33258 is a marker dye in Hoechst series. Hoechst is A live nuclear marker dye. Hoechst binds to the grooves in the DNA double strand, which tends to be A/ T-rich DNA strand. Although it binds to all nucleic acids, the A/ T-rich double strand DNA significantly enhances fluorescence intensity Therefore,Hoechst dye can be used for living cell labeling. The fluorescence intensity of Hoechst dye increases with the increase of pH of solution[1].

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]
Cellular Effect
Cell Line Type Value Description References
A2780 IC50
12 μM
Compound: HOE-33258
Antitumor activity of compound for 96-h exposure in A2780 human ovarian cell line
Antitumor activity of compound for 96-h exposure in A2780 human ovarian cell line
[PMID: 11170623]
A2780cisR IC50
9.5 μM
Compound: HOE-33258
Antitumor activity of compound for 96-h exposure in A2780cisR (acquired resistance to cisplatin) human ovarian cell line
Antitumor activity of compound for 96-h exposure in A2780cisR (acquired resistance to cisplatin) human ovarian cell line
[PMID: 11170623]
A549 IC50
≥ 31.2 μg/mL
Compound: 2
Cytotoxicity against human A549 cells assessed as reduction in cell survival after 24 hrs by resazurin assay
Cytotoxicity against human A549 cells assessed as reduction in cell survival after 24 hrs by resazurin assay
[PMID: 27301676]
AGS GI50
112 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against human AGS cells after 72 hrs by MTT assay
Cytotoxicity against human AGS cells after 72 hrs by MTT assay
[PMID: 21227551]
BEAS-2B IC50
≥ 31.2 μg/mL
Compound: 2
Cytotoxicity against human BEAS2B cells assessed as reduction in cell survival after 24 hrs by resazurin assay
Cytotoxicity against human BEAS2B cells assessed as reduction in cell survival after 24 hrs by resazurin assay
[PMID: 27301676]
Caco-2 GI50
84 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against human Caco2 cells after 72 hrs by MTT assay
Cytotoxicity against human Caco2 cells after 72 hrs by MTT assay
[PMID: 21227551]
CH1 IC50
26.5 μM
Compound: HOE-33258
Antitumor activity of compound for 96-h exposure in CH1 human ovarian cell line
Antitumor activity of compound for 96-h exposure in CH1 human ovarian cell line
[PMID: 11170623]
HeLa GI50
115.7 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against human HeLa cells after 72 hrs by MTT assay
Cytotoxicity against human HeLa cells after 72 hrs by MTT assay
[PMID: 21227551]
HeLa IC50
51.31 μM
Compound: H. 33258, Hoechst 33258
Cytotoxicity against human HeLa cells after 72 hrs by MTT assay
Cytotoxicity against human HeLa cells after 72 hrs by MTT assay
[PMID: 22959518]
HEp-2 GI50
132.5 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against human Hep2 cells after 72 hrs by MTT assay
Cytotoxicity against human Hep2 cells after 72 hrs by MTT assay
[PMID: 21227551]
HL-60 IC50
32.43 μM
Compound: H. 33258, Hoechst 33258
Cytotoxicity against human HL60 cells after 72 hrs by MTT assay
Cytotoxicity against human HL60 cells after 72 hrs by MTT assay
[PMID: 22959518]
MCF7 GI50
5.7 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against human MCF7 cells after 72 hrs by MTT assay
Cytotoxicity against human MCF7 cells after 72 hrs by MTT assay
[PMID: 21227551]
MDCK GI50
157.5 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against MDCK cells after 72 hrs by MTT assay
Cytotoxicity against MDCK cells after 72 hrs by MTT assay
[PMID: 21227551]
MIA PaCa-2 GI50
191.5 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against human MIAPaCa2 cells after 72 hrs by MTT assay
Cytotoxicity against human MIAPaCa2 cells after 72 hrs by MTT assay
[PMID: 21227551]
NCI-H358 GI50
91.7 x 10-6 mol/dm3
Compound: Hoechst 33258
Cytotoxicity against human NCI-H358 cells after 72 hrs by MTT assay
Cytotoxicity against human NCI-H358 cells after 72 hrs by MTT assay
[PMID: 21227551]
SK-OV-3 IC50
> 100 μM
Compound: HOE-33258
Antitumor activity of compound for 96-h exposure in SKOV-3 human ovarian cell line
Antitumor activity of compound for 96-h exposure in SKOV-3 human ovarian cell line
[PMID: 11170623]
U-937 IC50
15.42 μM
Compound: H. 33258, Hoechst 33258
Cytotoxicity against human U937 cells after 72 hrs by MTT assay
Cytotoxicity against human U937 cells after 72 hrs by MTT assay
[PMID: 22959518]
In Vitro

General Protocol
Preparation of Hoechst working solution
1.1 Preparation of the stock solution
Dissolve 10 mg of in 5 mL DMSO
Note: It is recommended to store the stock solution at 4℃ or -20℃ away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of Hoechst working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain final concentration 10 μg/mL Hoechst working solution.
Note: Please adjust the concentration of Hoechst working solution according to the actual situation.
1.Cell staining
2.1 Suspension cells（6-well plate）
a. Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time. The cell density is 1×106/mL.
b. Add 1 mL of working solution, and then incubate at room temperature for 3-10 minutes.
c. Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
d. Wash twice with PBS, 5 minutes each time.
e. Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslip from the medium and aspirate excess medium.
c. Add 100 μL of working solution, gently shake it to completely cover the cells,and then incubate at room temperature for 3-10 minutes.
d. Wash twice with medium, 5 minutes each time.Observation by fluorescence microscopy or flow cytometry.

Precautions
1. Please adjust the concentration of Hoechst working solution according to the actual situation.
2. This product is for R&D use only, not for drug, household, or other uses.
3. For your safety and health, please wear a lab coat and disposable gloves to operate.

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

Hoechst 33258 Related Antibodies
Molecular Weight

424.50

Formula

C25H24N6O
CAS No.

23491-44-3
Appearance

Solid

Color

Light yellow to yellow

Emission (Em)

451
Excitation (Ex)

356
SMILES

OC1=CC=C(C2=NC3=CC=C(C4=NC5=CC=C(N6CCN(C)CC6)C=C5N4)C=C3N2)C=C1
Shipping

Room temperature in continental US; may vary elsewhere.
Storage

-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; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
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
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

  • Molarity Calculator

  • Dilution Calculator

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

Mass
=
Concentration
×
Volume
×
Molecular Weight *

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

This equation is commonly abbreviated as: C1V1 = C2V2

Concentration (start)

C1

×
Volume (start)

V1

=
Concentration (final)

C2

×
Volume (final)

V2

In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 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

    This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
  • Protocol 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

    This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

Dosage

mg/kg

Animal weight
(per animal)

g

Dosing volume
(per animal)

μL

Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
DMSO +
% 
+
%
Tween-80 +
%
Saline
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration: mg/mL
Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).

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

The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation

Purity: 99.93%

SDS (393 KB)

COA (245 KB) LCMS (134 KB)

Handling Instructions (2659 KB)
Dyeing Example
  • Description: Hoechst 33258 can be used for detecting nuclei.
    Method: For cell staining.
    1. Fix, penetrate and seal cells first.
    2. Incubate cells with Hoechst 33258 (1:1000; 30 min; dark).
    3. Wash cells with PBS for five times and use a fluorescence microscope (ECLIPSE TE2000-5) for image.
  • Description: Hoechst 33258 can be used for detecting nuclei and staining nuclei of live and dead (apoptotic/necrotic).
    Method: For cell staining.
    1. Incubate cultured cells with 1 mL of the solution prepared with Hoechst 33258 (7.5 μg/mL; 37℃; 5% CO2; 30 min; 1×PBS).
    2. Use a Evos FLc Fluorescent Inverted Microscope for image.
  • Description: Hoechst 33258 can be used for detecting nuclei, it can be used in apoptosis detection assays.
    Method: For cell staining.
    1. Fix cells in paraformaldehyde after transfection and wash cells with PBS.
    2. Stain cells with Hoechst 33258 (30 min; 20℃; dark).
    3. Use a fluorescence microscope (Leica, Solms, Germany) for image.
  • Description: Hoechst 33258 can be used to visualize the nuclear DNA.
References

  • [1]. Wang XJ, et al. Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through inductionof apoptosis and autophagy. Bioorg Med Chem Lett. 2012 Oct 1;22(19):6297-300.  [Content Brief]

    [2]. Stolić I, et al. Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene. Eur J Med Chem. 2011 Feb;46(2):743-55.  [Content Brief]

Cell Assay
[2]

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.
References

  • [1]. Wang XJ, et al. Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through inductionof apoptosis and autophagy. Bioorg Med Chem Lett. 2012 Oct 1;22(19):6297-300.  [Content Brief]

    [2]. Stolić I, et al. Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene. Eur J Med Chem. 2011 Feb;46(2):743-55.  [Content Brief]

  • [1]. Wang XJ, et al. Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through inductionof apoptosis and autophagy. Bioorg Med Chem Lett. 2012 Oct 1;22(19):6297-300.

    [2]. Stolić I, et al. Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene. Eur J Med Chem. 2011 Feb;46(2):743-55.

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
DMSO 1 mM 2.3557 mL 11.7786 mL 23.5571 mL 58.8928 mL
5 mM 0.4711 mL 2.3557 mL 4.7114 mL 11.7786 mL
10 mM 0.2356 mL 1.1779 mL 2.3557 mL 5.8893 mL
15 mM 0.1570 mL 0.7852 mL 1.5705 mL 3.9262 mL
20 mM 0.1178 mL 0.5889 mL 1.1779 mL 2.9446 mL
25 mM 0.0942 mL 0.4711 mL 0.9423 mL 2.3557 mL
30 mM 0.0785 mL 0.3926 mL 0.7852 mL 1.9631 mL
40 mM 0.0589 mL 0.2945 mL 0.5889 mL 1.4723 mL
50 mM 0.0471 mL 0.2356 mL 0.4711 mL 1.1779 mL
60 mM 0.0393 mL 0.1963 mL 0.3926 mL 0.9815 mL
80 mM 0.0294 mL 0.1472 mL 0.2945 mL 0.7362 mL
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Hoechst 33258 Related Classifications

  • Cancer
  • Cancer Targeted Therapy Cancer Immunotherapy Cancer Metabolism and Metastasis
  • Others
  • Fluorescent Dye
Help & FAQs
  • Do most proteins show cross-species activity?

    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

Keywords:

Hoechst 3325823491-44-3bisBenzimide H 33258 H 33258Hoechst33258Hoechst-33258bisBenzimide H33258bisBenzimide H-33258H33258H 33258H-33258Fluorescent DyeHoechstDNAcellular dyeblue fluorescenceInhibitorinhibitorinhibit

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