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Real-time imaging of viable-apoptotic switch in GSNO-induced mouse thymocyte apoptosis
Dan-ying Lin1, Wan-yun Ma, Shao-jin Duan
1Key Laboratory for Atomic and Molecular Nanosciences of Education Ministry, Department of Physics, Tsinghua University, Beijing 100084, China.
Abstract:
Many scientists are focusing on the apoptotic-necrotic or the necrotic-apoptotic switch and its mechanism, but little attention has been paid to the viable-apoptotic switch. Most of the techniques and methods used for detecting apoptosis are performed on fixed samples, yielding static information of specific time points. We have studied the viable-apoptotic switch in S-nitrosoglutathione (GSNO)-induced mouse thymocyte apoptosis in real-time by means of a novel technique, intensified charge coupled device (ICCD)-based real-time fluorescence micro-imaging, coupled with Annexin V-FITC labeling for phosphatidylserine (PS) translocation in cell membrane. We have successfully recorded the initiating time points (mostly at 2 h) of the viable-apoptotic switch in GSNO-initiated apoptosis, as well as shown the real-time differences between living and apoptotic thymocytes. These findings suggest that NO is also a switch molecule for the conversion from viable to apoptotic cell. Thymocytes cotreated by N-G-monomethyl-L-arginine acetate salt (L-NMMA) provide further evidence for this suggestion, as well as for the suggestion that L-NMMA prolongs the early stage of thymocyte apoptosis rather than strongly blocks it.
Insights
Nitric oxide (NO) acts as a switch molecule in the viable-apoptotic switch of mouse thymocytes. This study used real-time imaging to observe the transition from living to apoptotic cells, revealing NO
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Apoptosis research often focuses on the switch between apoptotic and necrotic cell death pathways.
- Limited research exists on the transition from viable to apoptotic states.
- Current apoptosis detection methods typically use fixed samples, providing only static snapshots.
Purpose of the Study:
- To investigate the viable-apoptotic switch in S-nitrosoglutathione (GSNO)-induced mouse thymocyte apoptosis in real-time.
- To identify the role of nitric oxide (NO) in initiating this transition.
- To characterize the dynamic changes occurring during the shift from a living to an apoptotic state.
Main Methods:
- Utilized intensified charge coupled device (ICCD)-based real-time fluorescence micro-imaging.
- Employed Annexin V-FITC labeling to detect phosphatidylserine (PS) translocation.
- Studied mouse thymocytes treated with GSNO and N-G-monomethyl-L-arginine acetate salt (L-NMMA).
Main Results:
- Successfully recorded the initiation time points of the viable-apoptotic switch (around 2 hours) in GSNO-induced apoptosis.
- Demonstrated real-time differences between viable and apoptotic thymocytes.
- Observed that L-NMMA prolonged the early stages of thymocyte apoptosis rather than blocking it.
Conclusions:
- Nitric oxide (NO) functions as a switch molecule regulating the conversion of viable cells to apoptotic cells.
- Real-time imaging provides dynamic insights into the viable-apoptotic transition.
- L-NMMA's effect suggests a modulatory role in the early phase of apoptosis progression.
