DDAO

DDAO is a promising near-infrared (NIR) red fluorescent probewith tunable excitation wavelength (600-650nm) and longemission wavelength(λem=656nm). DDAO can be desiged for detection of the activities of different enzymes such asβ-galactosidase,sulfatase, proteinphosphatase2A,carboxylesterase 2, humanalbumin andesterases^[1]. IC50 & Target:Red fluorescent probe In Vitro: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 ℃ 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 ℃ 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.