Hoechst 34580 tetrahydrochloride (Synonyms: HOE 34580 tetrahydrochloride)

This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare Hoechst staining solution
(1) Prepare Hoechst dye stock solution: Use DMSO to dissolve the solid powder and prepare a 1-5 mg/mL Hoechst dye stock solution.
Note: Hoechst stock solution is recommended to be aliquoted and stored in the dark at -4°C or -20°C to avoid repeated freezing and thawing.
(2) Working solution preparation: Use preheated serum-free medium or buffer (such as HBSS or PBS) to dilute the stock solution and prepare a Hoechst working solution with a concentration of 1-10μg/mL.
Note: Please adjust the concentration of Hoechst working fluid according to the actual situation and prepare it immediately.
2. Cell staining
2.1 Suspension cells (taking 6-well plate as an example)
(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.
(2) Add 1 mL of Hoechst dye working solution and incubate at room temperature in the dark for 5-30 minutes.
(3) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.
(4) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through fluorescence microscopy or flow cytometry.
2.2 Adherent cells
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100μL of Hoechst dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 5-30 minutes.
(4) Aspirate away the dye working solution, wash the coverslip 2 to 3 times with culture solution, and observe with a fluorescence microscope.
3. Microscope detection: Hoechst 34580 can be excited by near-UV (375nm) or purple (405nm) laser, and the maximum excitation/emission light is 392⁄490nm respectively[1][2].
Precautions:
1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.
2) For your safety and health, please wear a lab coat and disposable gloves.
References:
[1].Karen E. Selph. Enumeration of marine microbial organisms by flow cytometry using near-UV excitation of Hoechst 34580-stained DNA.
[2]. Jonas Bucevičius,Gražvydas Lukinavičius , Rūta Gerasimaitė. The Use of Hoechst Dyes for DNA Staining and Beyond. Chemosensors 2018, 6(2), 18. Doi:org/10.3390/chemosensors6020018.