DDAO

DDAO is a promising near-infrared (NIR) red fluorescent probewith tunable excitation wavelength (600-650nm) and longemission wavelength(λem=656nm). DDAO can de 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:Guidelines (the following is our recommended protocol, which is provided as a guide only and should be modified to suit your specific needs).
1. Cell Samples Preparation^[1]:
1) HeLa cells are seeded into 15-mm confocal dishes at a density of 10⁵ cells in 1 mL of 1640 medium containing 10% fetal bovine serum.
2) HeLa cells are incubated at 37 ºC in a humidified incubator for 12 h.
2. Dye Stock Solution Preparation:
1) 9H-1,3-Dichloro-7-hydroxy-9,9-dimethylacridine-2-one (DDAO) is dissolved in dimethyl sulfoxide (DMSO) to prepare an AB1 stock solution with a concentration of 500 µM.
3. Dye Working Solution Preparation:
1) Take the AB1 stock solution and dilute it further with DMSO and 20 mM phosphate buffer (PB) at pH 7.4 until the final DMSO concentration is 30% (v/v) and the AB1 concentration is 30 µM.
4. Specific Staining Procedures
1) After 12 h of HeLa cell culture, the medium is replaced with fresh medium containing AB1 at a final concentration of 5 µM. The cells are incubated at 37 ºC for another 2 h.
2) Then, the cells are treated with H₂O₂ at a final concentration of 100 µM or Phorbol-12-myristate-13-acetate (HY-18739) (PMA) at 1 µg/mL for 30 min respectively. In the control experiment using N-acetylcysteine (NAC) as a scavenger of H₂O₂, the cells pre-loaded with the probe are incubated with NAC at a final concentration of 1 mM for 40 min before PMA stimulation.
3) After treatment, the cells are rinsed with PBS and fixed with mounting medium for 20 min.