ABSTRACT
Objective: To test the application of flow cytometry technique to detect the redox status with genetically encoded fluorescent probe roGFP2, to compare it with laser scanning confocal microscope (LSCM), and to demonstrate the diversity of cellular redox status in HeLa and Panc-1 cell populations. Methods: Time lapse imaging with LSCM was performed in single cell transfected with roGFP2 probe to detect the dynamic changes of 405 nm/488 nm ratio (405/488 ratio). Flowcytometry technique was also performed in HeLa cells transfected with roGFP2 probe to detect the dynamic alterations of 405/488 ratio. The global cell population was analyzed and the subpopulations of different redox status were dissected. Flowcytometry technique was further applied in Panc-1 cells with different CD24 or CD44 marker to detect the dynamic alterations of 405/488 ratio of roGFP2 and identify the different redox status. Results: Time lapse imaging with LSCM showed that 405/488 ratio of roGFP2 dramatically changed in response to H2O2 in dose and time dependent manner at a single cell level. When dozens of cells were chosen, the average ratio showed increased and dynamic trend. Compared to LSCM, flow cytometry could detect the average of 405/488 ratio of roGFP2 as well. Meanwhile, with the application of flow cytometry the cell population can be divided into three subpopulations based on 405/488 ratios, most in oxidized and medium redox status, few in reduced status. Using flow cytometry, CD24+/CD44+ Panc-1 cells, pancreatic cancer stem cells, can be found to have overall lower 405/488 ratio and more percentage of subpopulation of reduced status under resting and stress condition. Conclusion: Flow cytometry technique can be applied to detect roGFP2 and has advantage in application to show the overall as well as diverse redox status in cell population. Flow cytometry detection of cellular redox status with genetically encoded probe can be a useful tool in tumor cell biology and developmental biology research.
ABSTRACT
Objective·To test the application of flow cytometry technique to detect the redox status with genetically encoded fluorescent probe roGFP2,to compare it with laser scanning confocal microscope (LSCM),and to demonstrate the diversity of cellular redox status in HeLa and Pane-1 cell populations.Methods·Time lapse imaging with LSCM was performed in single cell transfected with roGFP2 probe to detect the dynamic changes of 405 nm/488 nm ratio (405/488 ratio).Flowcytometry technique was also performed in HeLa cells transfected with roGFP2 probe to detect the dynamic alterations of 405/488 ratio.The global cell population was analyzed and the subpopulations of different redox status were dissected.Flowcytometry technique was further applied in Panc-1 cells with different CD24 or CD44 marker to detect the dynamic alterations of 405/488 ratio of roGFP2 and identify the different redox status.Results·Thne lapse imaging with LSCM showed that 405/488 ratio of roGFP2 dramatically changed in response to H2O2in dose and time dependent manner at a single cell level.When dozens of cells were chosen,the average ratio showed increased and dynamic trend.Compared to LSCM,flow cytometry could detect the average of 405/488 ratio of roGFP2 as well.Meanwhile,with the application of flow cytometry the cell population can be divided into three subpopulations based on 405/488 ratios,most in oxidized and medium redox status,few in reduced status.Using flow cytometry,CD24+/CD44+ Panc-1 ceils,pancreatic cancer stem ceils,can be found to have overall lower 405/488 ratio and more percentage of subpopulation of reduced status under resting and stress condition.Conclusion·Flow cytometry technique can be applied to detect roGFP2 and has advantage in application to show the overall as well as diverse redox status in cell population.Flow cytometry detection of cellular redox status with genetically encoded probe can be a useful tool in tumor cell biology and developmental biology research.