Membrane Fluidity

Membrane fluidity was measured as previously described.¹⁹ Briefly, cells were incubated for 1 h at 37 °C in a humidified incubator with 10 μM 1-pyrenedecanoic acid (PDA) diluted in Live Cell Imaging Solution. Afterward, cells were incubated with the respective stimuli, and fluorescence was immediately measured with a Cytation 3 Cell Imaging Multi-Mode Reader equipped with the Gen5 Data Analysis Software (BioTek Instruments, Inc., Winooski, Vermont, USA). The signal was calculated as the ratio between ex/em. 344/470 nm for the PDA excimers and ex/em. 344/375 for the monomeric form. N-Acetylcysteine (10 mM NAC, Figure ​Figure66A,B) was included as antioxidant (assay control). Data are mean of minimum 6 independent experiments performed in quadruplicates.

Effect of ATXII on the membrane of THP-1 macrophages. (A, B) Membrane fluidity assay. Data are mean of n ≥ 6 independent experiments performed in quadruplicate. Symbols *# (p < 0.05) and ** ## (p < 0.01) indicate significant decrease (*) or increase (#) in comparison to controls Mann–Whitney test. (C, D) Morphological characterization of the cell membrane in control conditions or after incubation with 1 μM ATXII and/or 50 μM MβCD and in the presence of LPS 100 ng/mL. (E, F) CellMask signal quantification (RFU). Image analysis was performed on n ≥ 90 cells from 4 independent cell preparations. Symbols indicate difference *** p < 0.001 Student’s t-test. All concentrations are intended in [μM].