Genetically encoded red fluorescent pH ratiometric sensor: Application to measuring pH gradient abnormalities in cystic fibrosis cells Salto González, Rafael Girón González, María Dolores Paredes Martínez, José Manuel pH sensor Red Fluorescent protein Intracellular probes Lysosomal pH gradient Cystic fibrosis Lysosome-related diseases Genetically encoded probes to measure in vivo pH are challenging. They must be chloride insensitive and require normalization of the transfection efficiency. Furthermore, probes that emit in the red or far red are advisable to promote in vivo use. The mBeRFP D162T fluorescent protein presents two emission bands with different pH sensitivities. When the probe was cytosolic-expressed in HeLa as control cells and CFBE41o, an epithelial cell line that carries an F508del mutation in the CFTR transporter, the ratiometric measurement between both emission bands allows us to determine the pH, demonstrating that mBeRFP D162T can be used to accurately measure the cytosolic pH differences of these cell lines. Furthermore, we have located the sensor inside or outside the lysosomal membrane to investigate the lysosomal pH gradient. In HeLa cells, our probe detected pH gradient changes under conditions known to alter lysosomal pH. In the CFBE41o cells, which mimic cystic fibrosis disease, we observed a complete loss of lysosomal acidification. Using lumacaftor, a drug that restores functioning CFTR protein, partially brings back the pH gradient. In conclusion, mBeRFP D162T is a valuable tool for measuring in vivo intracellular pH values and lysosomal pH gradient dynamics in physiological or pathological conditions. 2024-04-03T06:33:47Z 2024-04-03T06:33:47Z 2024-03-21 info:eu-repo/semantics/article Sensors & Actuators: B. Chemical 410 (2024) 135673 [https://doi.org/10.1016/j.snb.2024.135673] https://hdl.handle.net/10481/90322 10.1016/j.snb.2024.135673 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess Attribution-NonCommercial-NoDerivatives 4.0 Internacional Elsevier