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 cytotoxic effect.

Physicochemical Properties

Molecular Formula	C40H39CLN2O8
Molecular Weight	711.199270486832
Exact Mass	710.239
CAS #	1448821-89-3
PubChem CID	162640791
Appearance	Purple to purplish red solid powder
LogP	7.7
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	10
Heavy Atom Count	51
Complexity	1290
Defined Atom Stereocenter Count	0
SMILES	O=C1C2=CC=CC=C2C2(C3=CC(Cl)=C(N(C4C=CC(O)=CC=4)C)C=C3OC3=CC4N(C(C)(C)CC(C)C=4C=C23)CCCC(=O)OCOC(=O)C)O1
InChi Key	AIRAPNPEUVLNDG-UHFFFAOYSA-N
InChi Code	InChI=1S/C40H39ClN2O8/c1-23-21-39(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/h6-7,9-10,12-15,17-20,23,45H,8,11,16,21-22H2,1-5H3
Chemical Name	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
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

1. The working solution for HKYellow-AM (6/12-mixture) is prepared as follows: dissolve 1 mg of HKYellow-AM (6/12-mixture) in 70 μL DMSO to create a 10 mM stock solution. Note: To prevent frequent freezing and thawing, it is advised that the stock solution be kept between -20°C and 80°C in the dark. 1.2 HKYellow-AM (6/12-mixture) working solution To create a 1–10 μM working solution, dilute the stock solution with either PBS or serum-free cell culture medium. Note: Please modify the working solution concentration of HKYellow-AM (6/12-mixed solution) based on the actual circumstances. 2. 2.1 Suspension cells (six-well plate) a. After centrifuging at 1000g for three to five minutes at 4°C, discard the supernatant. Wash for five minutes each time, twice, using PBS. 1×106/mL b is the cell density. After adding 1 mL of the working solution, let it sit at room temperature for five to thirty minutes. an. After centrifuging at 400 g for three to four minutes at 4 °C, discard the supernatant. D. Wash for five minutes each time, twice, using PBS. E. Using a fluorescence microscope or flow cytometry, resuspend the cells in PBS or serum-free cell culture media. Cells that adhere 2.2 a. Adherent cells are cultured on sterile coverslips. B. Lift the coverslip out of the culture media and use the aspirator to remove any extra. an. To fully cover the cells, add 100 μL of working solution, shake gently, and then incubate for five to thirty minutes at room temperature. D. Wash for five minutes twice using a medium setting. either flow cytometry monitoring or fluorescence microscopy.

References

[1]. Trends Cell Biol. 2007 Sep;17(9):422-7. doi: 10.1016/j.tcb.2007.07.009. Epub 2007 Sep 4. [2]. Diarylamine-based fluorogenic probes for detection of peroxynitrite. EP2809666B1. [3]. Diarylamine-based fluorogenic probes for detection of peroxynitrite. EP2809666B1.

Solubility Data

Solubility (In Vitro)

DMSO: 130 mg/mL (91.39 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 3.25 mg/mL (2.28 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 3.25 mg/mL (2.28 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.4061 mL	7.0304 mL	14.0607 mL
	5 mM	0.2812 mL	1.4061 mL	2.8121 mL
	10 mM	0.1406 mL	0.7030 mL	1.4061 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.