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[Fluorophore to detect hypochlorous acid in living cells] HySOx

  • High specificity to hypochlorous acid, and low reactivity with other reactive oxygen species (ROS).
  • Fast reactivity and slow photobleaching. It is suitable for time-lapse imaging.

Features

HySOx is a fluorescent probe that shows excellent selectivity against hypochlorous acid (HOCl). Hypochlorous acid is one of the various reactive oxygen species (ROS) including hydroxyradical (OH), superoxide (O2-・), hydrogen peroxide (H2O2), singlet oxygen (1O2), nitric oxide (NO), and peroxynitrite (ONOO).

Principle of the measurement

HySOx is almost non-fluorescent in neutral solutions. When HySOx reacts with hypochlorous acid, it generates a highly fluorescent product (with excitation maximum: 553 nm and emission maximum: 574 nm). 

Selectivity of HySOx

Figure 1. Reactivity of HySOx to various ROS
Figure 1. Reactivity of HySOx to various ROS

Fluorescence intensity of HySOx increases up to ~170-fold when it reacts with hypochlorous acid. Other ROS (superoxide, peroxynitrite, nitric oxide, singlet oxygen, and hydrogen peroxide) do not increase the fluorescent intensity of HySOx under identical conditions.

Measurement conditions
  • Fluorescent intensities of 5 µM HySOx were measured after the addition of various ROS in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1% DMF as a cosolvent.
  • Fluorescent intensities were measured at 574 nm, with excitation at 553 nm, slit width 2.5 nm, photon multiplier voltage 700V, using HITACHI F-2700 Fluorescence Spectrophotometer.
ROS generating conditions

HOCl: 5 µM NaOCl
OH: 50 µM Fe(ClO4)2, 100 µM H2O2
ONOO : 5 µM HOONO
NO: 5 µM NOC18
O2-・: 10 µM KO2
H2O2: 100 µM H2O2
PB: 0.1 M Sodium phosphate buffer (pH 7.4) as a control

Dose-dependent changes in the spectrum and intensity of HySOx fluorescence

Figure 2. Fluorescent spectrum changes of 5 µM HySOx solution in various concentrations of hypochlorous acid.
Figure 2. Fluorescent spectrum changes of 5 µM HySOx solution in various concentrations of hypochlorous acid.

The reaction of HySOx with hypochlorous acid saturates at the molar ratio of 1:2.
Inset, fluorescent intensities of HySOx against hypochlorous acid concentrations.

Measurement conditions
Fluorescent spectra of 5 µM HySOx were measured after the addition of hypochlorous acid (0-15 µM) in 0.1 M sodium phosphate buffer at pH 7.4 containing 0.1 % DMF as a cosolvent.

Inset, fluorescent intensities of 5 µM HySOx measured at 574 nm with 0-5 µM of incremental hypochlorous acid concentrations. The solutions were excited at 553 nm with slit width 2.5mm, and measured with photon multiplier voltage of 700V, using HITACHI F-2700 Fluorescence Spectrophotometer.

An application example
Observation of hypochlorous acid production from U937 cell during phagocytosis

  1. Induce differentiation of U937 cell, by incubation in the presence of 25 nM PMA for 24 h followed by 24 h incubation without PMA until observation.
  2. Dissolve HySOx 1 vial (20 µg) in 51.5 µL DMF to prepare 1 mM solution.
  3. Dilute the DMF stock solution with washing buffer or culture media to 5 µM cell staining solution.
  4. Add the cell staining solution to the dish and incubate at 37°C for 30 min.
  5. Remove the culture medium on the culture dish and wash twice with HBSS.
  6. Replace the buffer to new HBSS, and induce the phagocytosis by the addition of ZymosanA, and quickly start the time-lapse imaging.

※ Cells were treated with 50 μg/ml ZymosanA in this test.
※ Microscope: Leica DMI 6000 CS, objective lens: 40×
※ In our test, the fluorescent signal appeared in 10 minutes, and increased after 30 minutes.

Figure 4. Hypochlorous acid production during phagocyte of ZymosanA by U937 cell
Figure 3. Hypochlorous acid production during phagocyte of ZymosanA by U937 cell

(Left) Immediately after an addition of ZymosanA to U937 cell
(Middle) 10 minutes after the addition of ZymosanA. U937 cell engulfed the ZymosanA and started to show red fluorescent signal (arrow).
(Right) 30 minutes after the addition of ZymosanA. Phagocytosis was completed. Strong red fluorescent signal indicates the production of hypochlorous acid in the cell.

YouTube

U937 cell
https://www.youtube.com/watch?v=PAyBEwesV-Y

RAW264.7 cell
https://www.youtube.com/watch?v=HFGghuqAwCQ

Reference
Ishida T, Suzuki S, Lai CY, Yamazaki S, Kakuta S, Iwakura Y, Nojima M, Takeuchi Y, Higashihara M, Nakauchi H, Otsu M.
Stem Cells. 2016 Oct 18. doi: 10.1002/stem.2524.
Suguru Kenmoku, Yasuteru Urano, Hirotatsu Kojima and Tetsuo Nagano
J. Am. Chem. Soc., 2007, 129 (23), pp 7313-7318 doi: 10.1021/ja068740g, publication date (Web): May 17, 2007
Price
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GC3006-01 HySOx 20μg×5 $498 Download
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