1. Vimentin regulates intracellular trafficking in B cells.
Vimentin regulates intracellular trafficking in B cells. (A–F) Primary B cells from WT and Vim−/− mice were stimulated using Alexa647-conjugated anti-IgM for 30 min. The cells were then fixed onto poly-lysine–coated coverslips and permeabilised. Next, the cells were labelled for LAMP1 suing anti-LAMP1 antibody and the corresponding secondary antibody. The samples were imaged using confocal microscopy. Single cells from the confocal images were then cropped and analysed individually using Matlab for cluster analysis. (A, B) The cluster number, asymmetry, and cluster half-width of antigen and (C, D) LAMP1 clusters were quantitated. Scale bar = 2 μm. (E, F) The degree of colocalisation between LAMP1 and antigen was analysed using Pearson's correlation and Manders coefficient. Scale bar = 5 μm. (G) Primary B cells from WT and Vim−/− mice were incubated with microspheres coated with biotinylated anti-IgM for 30 min on ice. Next, the cells were incubated at 37°C for up to 60 min. The cells were fixed at the end of the incubation and the remaining surface microspheres were detected using a fluorescent streptavidin. The samples were analysed using flow cytometry and the mean fluorescence intensity (MFI) of streptavidin was quantified. Data are representative of three independent experiments. (H) Primary B cells from WT and Vim−/− mice were incubated with microspheres coated with biotinylated anti-IgM and Eα peptide for 30 min at 37°C. Next, the cells were incubated at 37°C for up to 5 h. The cells were fixed at the end of the incubation and the MHC-II–Eα complexes were detected on the surface of the cells using a specific antibody and the corresponding secondary antibody. The samples were analysed using flow cytometry and the population of cells exhibiting high MFI (gated population) was quantified. Data are representative of three independent experiments.
Carlson Tsui et al. LSA 2018;1:e
© 2018 Tsui et al.