HKYellow-AM (6/12-mixture)

• CAT Number: I045486
• CAS Number: 1448821-89-3
• Molecular Formula: C80H78Cl2N4O16
• Molecular Weight: 1422.40
• Purity: ≥95%
• Synonyms: acetyloxymethyl 4-[8′-chloro-9′-(4-hydroxy-N-methylanilino)-2′,2′,4′-trimethyl-3-oxospiro[2-benzofuran-1,6′-3,4-dihydrochromeno[3,2-g]quinoline]-1′-yl]butanoate;acetyloxymethyl 4-[10′-chloro-9′-(4-hydroxy-N-methylanilino)-2′,2′,4′-trimethyl-3-oxospiro[2-benzofuran-1,12′-3,4-dihydrochromeno[2,3-h]quinoline]-1′-yl]butanoate
• InChI: InChI=1S/2C40H39ClN2O8/c1-23-21-39(3,4)43(18-8-11-35(46)49-22-48-24(2)44)37-27(23)16-17-33-36(37)40(29-10-7-6-9-28(29)38(47)51-40)30-19-31(41)32(20-34(30)50-33)42(5)25-12-14-26(45)15-13-25;1-23-21-39(3,4)43(16-8-11-37(46)49-22-48-24(2)44)33-19-35-30(17-28(23)33)40(29-10-7-6-9-27(29)38(47)51-40)31-18-32(41)34(20-36(31)50-35)42(5)25-12-14-26(45)15-13-25/h6-7,9-10,12-17,19-20,23,45H,8,11,18,21-22H2,1-5H3;6-7,9-10,12-15,17-20,23,45H,8,11,16,21-22H2,1-5H3
• InChIKey: WWDXCCHKVDGIDU-UHFFFAOYSA-N
• SMILES: CC1CC(N(C2=C1C=CC3=C2C4(C5=CC=CC=C5C(=O)O4)C6=CC(=C(C=C6O3)N(C)C7=CC=C(C=C7)O)Cl)CCCC(=O)OCOC(=O)C)(C)C.CC1CC(N(C2=CC3=C(C=C12)C4(C5=CC=CC=C5C(=O)O4)C6=CC(=C(C=C6O3)N(C)C7=CC=C(C=C7)O)Cl)CCCC(=O)OCOC(=O)C)(C)C

HKYellow-AM (6/12-mixture) is a yellow fluorescent probe that can detect ONOO- in living cells and tissues with high selectivity and sensitivity without cytotoxicity[2].
1. Preparation of HKYellow-AM (6/12-mixture) working solution
1.1 Preparation of the stock solution
Dissolve 1 mg HKYellow-AM (6/12-mixture) in 70 μL DMSO to obtain 10 mM of stock solution.
Note: It is recommended to store the stock solution at -20℃ -80℃ away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of HKYellow-AM (6/12-mixture) working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of working solution.
Note: Please adjust the concentration of HKYellow-AM (6/12-mixture) working solution according to the actual situation.
2. Cell staining
2.1 Suspension cells (6-well plate)
a. Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.The cell density is 1×106/mL
b. Add 1 mL of working solution, and then incubate at room temperature for 5-30 minutes.
c. Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
d. Wash twice with PBS, 5 minutes each time.
e. Resuspend cells with serum-free cell culture medium or PBS.Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslip from the medium and aspirate excess medium.
c. Add 100 μL of working solution, gently shake it to completely cover the cells,and then incubate at room temperature for 5-30 minutes.
d. Wash twice with medium, 5 minutes each time.Observation by fluorescence microscopy or flow cytometry.

Reference

[1]. Trends Cell Biol. 2007 Sep;17(9):422-7. doi: 10.1016/j.tcb.2007.07.009. Epub 2007 Sep 4.
 [Content Brief]

[2]. Yang D, et, al. Diarylamine-based fluorogenic probes for detection of peroxynitrite. EP2809666B1.

[3]. Yang D, et, al. Diarylamine-based fluorogenic probes for detection of peroxynitrite. EP2809666B1.