HKSOX-1m (5/6-mixture) (hydrobromide)

• CAT Number: I045008
• Molecular Formula: C49H37Br2F10N2O10PS2
• Molecular Weight: 1258.72
• Purity: ≥95%
• Synonyms: triphenyl-[4-[4-[2′,4′,5′,7′-tetrafluoro-1-oxo-3′,6′-bis(trifluoromethylsulfonyloxy)spiro[2-benzofuran-3,9′-xanthene]-5-carbonyl]piperazin-1-yl]butyl]phosphanium;triphenyl-[4-[4-[2′,4′,5′,7′-tetrafluoro-3-oxo-3′,6′-bis(trifluoromethylsulfonyloxy)spiro[2-benzofuran-1,9′-xanthene]-5-carbonyl]piperazin-1-yl]butyl]phosphanium;dibromide;dihydrobromide
• InChI: InChI=1S/2C49H36F10N2O10PS2.4BrH/c50-37-27-35-41(39(52)43(37)70-73(64,65)48(54,55)56)68-42-36(28-38(51)44(40(42)53)71-74(66,67)49(57,58)59)47(35)34-19-18-29(26-33(34)46(63)69-47)45(62)61-23-21-60(22-24-61)20-10-11-25-72(30-12-4-1-5-13-30,31-14-6-2-7-15-31)32-16-8-3-9-17-32;50-37-27-35-41(39(52)43(37)70-73(64,65)48(54,55)56)68-42-36(28-38(51)44(40(42)53)71-74(66,67)49(57,58)59)47(35)34-26-29(18-19-33(34)46(63)69-47)45(62)61-23-21-60(22-24-61)20-10-11-25-72(30-12-4-1-5-13-30,31-14-6-2-7-15-31)32-16-8-3-9-17-32;;;;/h2*1-9,12-19,26-28H,10-11,20-25H2;4*1H/q2*+1;;;;/p-2
• InChIKey: BYOVJHJLFIAGRN-UHFFFAOYSA-L
• SMILES: C1CN(CCN1CCCC[P+](C2=CC=CC=C2)(C3=CC=CC=C3)C4=CC=CC=C4)C(=O)C5=CC6=C(C=C5)C7(C8=CC(=C(C(=C8OC9=C(C(=C(C=C97)F)OS(=O)(=O)C(F)(F)F)F)F)OS(=O)(=O)C(F)(F)F)F)OC6=O.C1CN(CCN1CCCC[P+](C2=CC=CC=C2)(C3=CC=CC=C3)C4=CC=CC=C4)C(=O)C5=CC6=C(C=C5)C(=O)OC67C8=CC(=C(C(=C8OC9=C(C(=C(C=C79)F)OS(=O)(=O)C(F)(F)F)F)F)OS(=O)(=O)C(F)(F)F)F.Br.Br.[Br-].[Br-]

HKSOX-1m (5/6-mixture) hydrobromide is a O2 fluorescent probe for mitochondria-targeting (Ex/Em=509/534nm; green), exhibiting excellent selectivity and sensitivity toward O2 over a broad range of pH, strong oxidants, and abundant reductants found in cells[1].
HKSOX-1m (5/6-mixture) hydrobromide (10 μM) sensitively captures signals for basal and Antimycin A (1 μM)-stimulated mitochondrial O2 in differentiated human THP-1 cells, at very low laser power output (1% intensity at Ex 514 nm on Zeiss LSM 510 Meta)[1].
1. Preparation of HKSOX-1m (5/6-mixture) hydrobromide working solution
1.1 Preparation of the stock solution
Dissolve 1 mg HKSOX-1m (5/6-mixture) hydrobromide in 79 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 HKSOX-1m (5/6-mixture) hydrobromide 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 HKSOX-1m (5/6-mixture) hydrobromide 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]. Jun Jacob Hu, et al. Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo. J Am Chem Soc . 2015 Jun 3;137(21):6837-43.
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