Hoechst 33258 3HCl is a widely used nucleic acid stain which can bind to minor-groove of dsDNA and emit blue fluorescence.

The solid could be stored in dark at -20 ℃. The stock solution should be stored at 4°C in the dark for up to 6 months.

Make a stock solution of Hoechst 33258 at 1 mg/ml in ddH₂O. The stock solution can be stored at 4°C in the dark for up to 6 months

For cell suspension

1. Incubate cell suspensions (0.5-2.0 × 10⁶ cells/ml, in PBS) with Hoechst 33258 working solution at 1-10 μg/mL for 10 min. Usually the concentration of working solution at 1 μg/mL is enough (For example, add 1 μL Hoechst 33258 stock solution in 1 mL cell suspension.).

Note: Hoechst 33258 can stain nuclei of both live and dead cells.

2. Apply samples to cytospin chambers and centrifuge at 500 rpm for 2 minutes. After air-drying, place glass coverslips over cells to reduce light diffraction (no mounting gel necessary) and observe cells in a fluorescence microscope equipped with a DAPI filter, using a 60X objective.{{Kasibhatla S, Amarante-Mendes GP, Finucane D, Brunner T, Bossy-Wetzel E, Green DR. Staining of suspension cells with hoechst 33258 to detect apoptosis. CSH Protoc. 2006 Aug 1;2006(3):pdb.prot4492. doi: 1

Hoechst 33258 3HCl is a widely used nucleic acid stain which can bind to minor-groove of dsDNA and emit blue fluorescence. It is similar to Hoechst 33342. Hoechst 33258 is excited at 352 nm and emits at 461 nm.