DDAO

Guide (The following is our recommended protocol. This protocol is provided as a guideline only and should be modified according to your specific needs).
1. Cell Sample Preparation^[1]:
1. Seed HeLa cells in a confocal culture dish at a density of 10⁵ cells and add 1 mL of 1640 medium containing 10% fetal bovine serum.
2. Incubate HeLa cells in a 37 oC incubator for 12 h.
2. Dye Stock Solution Preparation:
1. Dissolve 9H-1,3-Dichloro-7-hydroxy-9,9-dimethylacridine-2-one (DDAO) in dimethyl sulfoxide (DMSO) to prepare an AB1 stock solution at a concentration of 500 μM.
3. Dye Working Solution Preparation:
1. Take the AB1 stock solution and further dilute it with DMSO and 20 mM pH 7.4 phosphate buffer (PB) until the final DMSO concentration is 30% (v/v) and the AB1 concentration is 30 μM.
4. Specific Staining Procedure
1. After culturing HeLa cells for 12 h, replace the medium with fresh medium containing AB1 at a final concentration of 5 μM. Continue incubating the cells at 37 oC for 2 h.
2. Then treat the cells for 30 min with either H₂O₂ at a final concentration of 100 μM or Phorbol-12-myristate-13-acetate (HY-18739) (PMA) at a concentration of 1 μg/mL. In control experiments using N-acetylcysteine (NAC) as an H₂O₂ scavenger, incubate probe-preloaded cells with NAC at a final concentration of 1 mM for 40 min prior to PMA stimulation.
3. After treatment, rinse cells with PBS and mount with mounting medium for 20 min.

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