Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis
Abstract
:1. Introduction
2. Results
2.1. Phagocytosis Assay
2.2. Characterization of POS Purity Using a Flow-Cytometry-Based Method Targeting Specific ROSs and COS Markers
2.3. Study of POS Stability under Several Stress Conditions
3. Discussion
4. Materials and Methods
4.1. Cell Media, Solvents, and Reagents
4.2. RPE Cells Preparation
4.3. Phagocytosis Assay
4.3.1. POS Batches
4.3.2. POS Conjugation with pHrodo™ Dye
4.3.3. Phagocytosis Assay
4.4. POS Sample Preparation
4.4.1. Heat-Stress-Treated Samples
4.4.2. Light-Stress-Treated Samples
4.4.3. In-House POS Extraction and Purification
4.5. Characterization of POS Population Using Flow Cytometry
4.5.1. POS Antibody Staining
4.5.2. Flow Cytometry
4.5.3. Analysis of Intracellular and Extracellular Soluble Proteins via HPLC/UV
4.5.4. Characterization of POSs Using Microscopy
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Liang, H.; Wu, Q.; Guo, X.V.; Chan, L.; Mao, T.; Stella, C.; Guilbaud, A.; Camperi, J. Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis. Int. J. Mol. Sci. 2023, 24, 12889. https://doi.org/10.3390/ijms241612889
Liang H, Wu Q, Guo XV, Chan L, Mao T, Stella C, Guilbaud A, Camperi J. Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis. International Journal of Molecular Sciences. 2023; 24(16):12889. https://doi.org/10.3390/ijms241612889
Chicago/Turabian StyleLiang, Haoqian, Qiang Wu, Xinzheng Victor Guo, Linda Chan, Tin Mao, Cinzia Stella, Axel Guilbaud, and Julien Camperi. 2023. "Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis" International Journal of Molecular Sciences 24, no. 16: 12889. https://doi.org/10.3390/ijms241612889