研究目的
Investigating the chemical dynamics of reactive oxygen species (ROS) and reactive nitrogen species (RNS) within the phagosome to understand pathogen destruction mechanisms.
研究成果
The study successfully maps phagosomal HOCl production and pH changes using a DNA-based reporter, revealing a burst of HOCl upon phagosome acidification. This method provides insights into phagosome maturation and MPO activity, with potential applications in studying immune-mediated inflammatory diseases and pathogen resistance mechanisms.
研究不足
The study is limited by the irreversible nature of the HOCl reporter, which may consume a fraction of the total HOCl generated. Additionally, the DNA scaffold's stability under harsh conditions and the potential for photobleaching over extended imaging periods are considerations.
1：Experimental Design and Method Selection:
The study employs a DNA-based fluorescent reporter, cHOClate, designed to simultaneously image hypochlorous acid (HOCl) and pH within phagosomes. The reporter integrates a HOCl-sensitive fluorophore, a pH-sensitive fluorophore, an internal standard for ratiometric quantitation, and a phagosome-targeting function.
2：Sample Selection and Data Sources:
The study uses live cells, including macrophages, neutrophils, and monocytes, to observe phagosomal HOCl production and pH changes.
3：List of Experimental Equipment and Materials:
The study utilizes a Fluoromax-4 spectrophotometer for fluorescence measurements, an IX-83 microscope for imaging, and various chemical reagents for ROS and RNS generation.
4：Experimental Procedures and Operational Workflow:
Cells are incubated with cHOClate, and fluorescence imaging is performed to monitor HOCl and pH changes in phagosomes over time. Pharmacological inhibitors are used to validate the specificity of HOCl detection.
5：Data Analysis Methods:
Fluorescence intensities are measured and ratios (R/B for HOCl and G/B for pH) are calculated to quantify changes. Image analysis is performed using ImageJ software.
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