研究目的
To develop a method for dynamic and reversible visual analysis of mitochondrial redox (H2O2/GSH) in single cells using a microfluidic device and a reversible fluorescent probe, and to explore changes under thermal and combined thermal-drug stimulation.
研究成果
The developed method successfully enables real-time fluorescent imaging analysis of mitochondrial redox changes in single cells. It reveals dynamic responses to thermal and combined stimuli, correlates redox changes with apoptosis, and highlights cellular heterogeneity. This approach provides a convenient platform for studying cellular redox and responses to external stimuli, with potential applications in therapeutic evaluation.
研究不足
The method may have limitations in handling very low concentrations of mitochondrial H2O2/GSH due to their high activity and easy transformation. The microfluidic device requires precise control of temperature and flow, which could introduce variability. The study focuses on specific cell lines and stimuli, and may not be generalizable to all cell types or conditions.
1：Experimental Design and Method Selection:
The study uses a microfluidic device integrated with a reversible fluorescent probe (Cy-O-ebselen) for online culture, labeling, and dynamic fluorescent imaging of mitochondrial redox changes. The device includes a microchip, gravity drive pump, CO2-control, temp-control, and laser scanning confocal microscope unit.
2：Sample Selection and Data Sources:
MCF-7 and MCF-10A cell lines were used, purchased from Nanjing Keygen Biological Technology Co., LTD. Cells were cultured and treated with thermal stimuli (42-46°C) or combined thermal/NEM stimuli.
3：List of Experimental Equipment and Materials:
Equipment includes a Leica TCS SP5 laser scanning confocal microscope, gravity drive pump, microfluidic chip, and various reagents such as Hoechst 33258, Annexin V-FITC apoptosis kit, NEM, and the Cy-O-Eb probe. Materials include culture media, buffers, and chemicals from suppliers like Gibco, Beyotime, Santa Cruz, CST, and Sigma.
4：Experimental Procedures and Operational Workflow:
The microfluidic chip was sterilized and coated with polylysine. Cells were introduced via gravity drive, incubated with the probe, and subjected to thermal stimuli. Fluorescent imaging was performed at specified intervals using confocal microscopy. Additional assays included nuclear staining, flow cytometry, and Western blot for apoptosis evaluation.
5：Data Analysis Methods:
Fluorescent intensity was quantified from images. Flow cytometry data were analyzed for apoptosis rates, and Western blot results were assessed using gel imaging and gray analysis software.
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