Fig. 4. Calcium signalling in lipid rafts. CHO cells transfected with CD14 and TLR4 were incubated for 30 minutes in medium supplemented with 2 µM X-Rhod-1 1/AM and 25 mg/l Pluronic F-127. LPS was added at zero time, resulting in the production of Ca²⁺, which was detected as an increase in X-Rhod 1 fluorescence. Lipid rafts were visualised using Cy5-cholera-toxin (A), TLR4 was visualised using OG-TLR4 (B); Ca²⁺ signal was visualised by the fluorescence of X-Rhod-1 (C); merged image (D). (E) Ca²⁺ responses monitored by X-Rhod-1 fluorescence in cells expressing clustered (cell 1, solid line) or not clustered (cell 2, dashed line) TLR4 molecules. (F) Mobility of TLR4 measured by FRAP in cells exhibiting clusters (cell 1, solid line), as well as diffuse (cell 2, dashed line) TLR molecules.