Intracellular H2O2 was visualized using HYDROP (Goryo Chemical Inc.) as described previously [13 (link)]. Briefly, cells in glass-bottom dishes (Matsunami Glass) were cultured in RPMI 1640 with 50 μM H2O2 for 1 h. After washing out the H2O2 twice with RPMI 1640, the cells were treated with 2.5 μM HYDROP in RPMI 1640 at 37 °C for 20 min. Then, the cells were washed twice with RPMI 1640. Fluorescence images were obtained using a BZ-8000 fluorescence microscope (KEYENCE) with a GFP-BP filter. ImageJ software was used to measure fluorescence intensity.
Hydrop
Manufactured by Matsunami
Sourced in Japan
HYDROP is a laboratory equipment designed for water treatment and purification applications. It functions as a water filtration and purification system, capable of removing impurities and contaminants from water samples.
Automatically generated - may contain errors
3 protocols using hydrop
1
Visualizing Intracellular Hydrogen Peroxide
2
Visualization of Intracellular H2O2 Using HYDROP
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Internal H2O2 was visualized using HYDROP™ (Goryo Chemical Inc., Hokkaido, Japan) as previously described.17 Briefly, cells in glass‐bottomed dishes (Matsunami Glass Ind., Ltd., Osaka, Japan) were cultured in RPMI 1640 with or without 50 μmol/L H2O2 for 10 min, 30 min, 1 h, and 2 h. The cultured cells were washed with noncontaining H2O2 RPMI 1640 twice to remove the H2O2 from the medium, and subjected to treatment with 2.5 μmol/L HYDROP™ in RPMI 1640 at 37ºC for 20 min. Subsequently, the cells were washed with RPMI 1640 twice, and fluorescence images were obtained using a BZ‐8000 fluorescence microscope (Keyence Corporation, Osaka, Japan). The ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/ , 1997‐2012) was used to measure fluorescence intensity.
3
Membrane Potential and H2O2 Imaging
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Plasma membrane potential was measured using bis-(1,3-dibutylbarbituric acid) trimethine oxonol sodium salt (DiBAC 4 (3)) (Dojindo, Kumamoto, Japan), and internal H 2 O 2 was visualized using HYDROP (Goryo Chemical Inc., Hokkaido, Japan) as reported previously. 19 Cells on glass-bottomed dishes (Matsunami Glass Ind., Ltd., Osaka, Japan) were treated with 5 mM DiBAC 4 (3) for 10 min at 37°C or with 2.5 mM HYDROP for 20 min at 37°C. After incubation, dyes were washed out and fluorescence images were obtained using BZ-8000 fluorescence microscope (KEYENCE Corporation, Osaka, Japan). Fluorescence intensities were measured using ImageJ (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA; http://rsb.info.nih.gov/ij/, 1997-2012.) Table 1. Primer sequences used in this study.
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