Journal: Bio-protocol
Article Title: X-Ray Photon Correlation Spectroscopy, Microscopy, and Fluorescence Recovery After Photobleaching to Study Phase Separation and Liquid-to-Solid Transition of Prion Protein Condensates
doi: 10.21769/BioProtoc.5277
Figure Lengend Snippet: (A) HEK293 cells transfected with PrP C -YFP-GPI or YFP-GPI for 48 h were exposed to 300 μM CuCl 2 in the cell media for 1 h or not. After that, two different sets of bleaching regions were performed for each of the experimental conditions: (i) bleaching was performed in isolated membranes by drawing a region of interest (ROI) at the cell surface of a membrane facing the cell medium, and (ii) an ROI was drawn at the cell–cell interfaces in which the membranes of two adjacent cells were in contact. FRAP experiments were collected for each condition followed by analysis and processing of data that included background correction and normalization as explained in section M. (B, C) FRAP recovery of PrP C -YFP-GPI or YFP-GPI at cell–cell interfaces vs. individual cell membranes upon Cu 2+ treatment or not. Inset show examples of regions before bleaching, directly after bleaching (bleach), and post-fluorescence recovery. Times to half-recovery (t 1/2 ) are indicated inside rectangles, and mobile phases (F ∞ ) in percentage are indicated next to the curves. The experiment was conducted on a confocal spinning disk microscope coupled with a stage-top incubator (37 °C; 5% CO 2 and humidity control). ROIs were bleached using a 488 nm laser at 100% laser intensity (2 loops, 0.4 s duration), and a timelapse was collected with frames acquired every 200 ms for 2 min (10% 488 nm laser). Scale bars are 1 μm. Graphs show the mean ± S.E.M. of recovery curves. Panels B and C are reprinted/adapted from Do Amaral et al. [12] Science Advances , DOI: 10.1126/sciadv.adi7347. Some rights reserved; exclusive licensee American Association for the Advancement of Science (AAAS). Distributed under a Creative Commons Attribution Non-Commercial License 4.0 (CC BY-NC 4.0) http://creativecommons.org/licenses/by-nc/4.0/ .
Article Snippet: Confocal spinning disk inverted microscope (Nikon, model: Eclipse-Ti CSU-X) equipped with a 60× objective (oil-immersion, 60×, plan Apo, oil, NA 1.4, Nikon), perfect focus system (PFS), and coupled with temperature, humidity, and CO 2 hypoxia incubator (On Stage Incubator, Okolab) 4.
Techniques: Transfection, Isolation, Membrane, Fluorescence, Microscopy, Control
![(A) HEK293 cells transfected with PrP C -YFP-GPI or YFP-GPI for 48 h were exposed to 300 μM CuCl 2 in the cell media for 1 h or not. After that, two different sets of bleaching regions were performed for each of the experimental conditions: (i) bleaching was performed in isolated membranes by drawing a region of interest (ROI) at the cell surface of a membrane facing the cell medium, and (ii) an ROI was drawn at the cell–cell interfaces in which the membranes of two adjacent cells were in contact. FRAP experiments were collected for each condition followed by analysis and processing of data that included background correction and normalization as explained in section M. (B, C) FRAP recovery of PrP C -YFP-GPI or YFP-GPI at cell–cell interfaces vs. individual cell membranes upon Cu 2+ treatment or not. Inset show examples of regions before bleaching, directly after bleaching (bleach), and post-fluorescence recovery. Times to half-recovery (t 1/2 ) are indicated inside rectangles, and mobile phases (F ∞ ) in percentage are indicated next to the curves. The experiment was conducted on a confocal spinning disk <t>microscope</t> coupled with a stage-top incubator (37 °C; 5% CO 2 and humidity control). ROIs were bleached using a 488 nm laser at 100% laser intensity (2 loops, 0.4 s duration), and a timelapse was collected with frames acquired every 200 ms for 2 min (10% 488 nm laser). Scale bars are 1 μm. Graphs show the mean ± S.E.M. of recovery curves. Panels B and C are reprinted/adapted from Do Amaral et al. [12] Science Advances , DOI: 10.1126/sciadv.adi7347. Some rights reserved; exclusive licensee American Association for the Advancement of Science (AAAS). Distributed under a Creative Commons Attribution Non-Commercial License 4.0 (CC BY-NC 4.0) http://creativecommons.org/licenses/by-nc/4.0/ .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1588/pmc12021588/pmc12021588__BioProtoc-15-8-5277-g006.jpg)