Hoechst 33258 is a blue to blue-green fluorescent live cell dye that can label DNA. Hoechst 33258 can specifically bind to the minor groove of DNA (and tends to bind to A/T-rich DNA), resulting in a significant increase in fluorescence intensity. Hoechst 33258 can cross the cell membrane and cause changes in DNA structure, such as G2/M phase arrest. Hoechst 33258 can bind to live or fixed cells, and the fluorescence intensity increases with increasing solution pH. As a DNA-specific probe, Hoechst 33258 can be used to detect DNA content, analyze cell cycle, etc. The excitation wavelength of Hoechst 33258 is 350-365 nm, and the emission wavelength is 460-490 nm^[1][2][3].

Guide (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Hoechst 33258 has two binding modes: Type 1 and Type 2. Type 1 binds at low concentrations, has high fluorescence quantum yield, and has an emission peak at 460 nm; Type 2 binds at higher concentrations, resulting in fluorescence quenching, with an emission peak at around 490 nm^[1].
The IC₅₀ values ??of Hoechst 33258 for HeLa, HL60, and U937 cells are 51.31 μM, 32.43 μM, and 15.42 μM, respectively^[2].

For the usage of Hoechst 33258, refer to ^[1]
1 Preparation of Hoechst working solution:
1.1 Preparation of stock solution Prepare Hoechst 33258 powder into 1-10 mM stock solution, and stir in dark place until completely dissolved.
Note: The dye is light sensitive, so the preparation process needs to be kept away from light.
1.2 Working solution dilution
Before use, dilute the stock solution with PBS or cell culture medium to the working concentration, usually 5-10 μg/mL (cell staining) or 1-50 μM (DNA binding study).
Note: Prepare immediately before use to avoid fluorescence quenching.
2 Adherent cell staining steps:
Cells are seeded in culture plates/dishes and cultured to the logarithmic growth phase. If cells need to be fixed, fix them with 4% paraformaldehyde at room temperature for 10-15 min, and wash twice with PBS for 5 minutes each time.
2.1 Add Hoechst 33258 working solution and incubate at 37°C in the dark for 10-30 min (adjust the time according to the cell type to avoid over-staining).
2.2 For live cell staining, add directly to the culture medium and incubate without fixation; for dead cells, fix first and then stain.
2.3 Wash the cells 2-3 times with PBS or serum-free medium to remove unbound dye and reduce background fluorescence.
3 Suspended cell staining steps:
3.1 Collect cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant.
3.2 Resuspend the cells with PBS, add Hoechst 33258 working solution, and incubate at room temperature in the dark for 15-20 min.
3.3 Resuspend after centrifugation and washing for flow cytometry or fluorescence microscopy detection.
4 Detection channels and tools:
4.1 Excitation wavelength (Ex): 350-365 nm (ultraviolet or near-ultraviolet light).
4.2 Emission wavelength (Em): 460-490 nm (blue to blue-green fluorescence).
4.3 Detection tools:
Fluorescence microscope: equipped with UV excitation filter (such as DAPI channel) to observe the intracellular DNA distribution.
Flow cytometer: Use UV laser (355 nm) and 450/50 nm bandpass filter to detect cell cycle or DNA content.
Microplate reader: When detecting in multi-well plate, select UV excitation and blue emission channels for high-throughput DNA quantification.
5 Precautions:
5.1 The concentration needs to be adjusted according to the cell type and experimental purpose. High concentration (>50 μM) may cause DNA precipitation (e.g., precipitation when D/P>0.4).
5.2 Short-term incubation (<30 min) has low cytotoxicity, and long-term incubation requires verification of cell viability.

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

* 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.

• [1]. Stokke T, et al. Multiple binding modes for Hoechst 33258 to DNA. J Histochem Cytochem. 1985 Apr;33(4):333-8.  [Content Brief]

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

  [3]. Pjura PE, et al. Binding of Hoechst 33258 to the minor groove of B-DNA. J Mol Biol. 1987 Sep 20;197(2):257-71.  [Content Brief]