Fig. 1.
Arrangement of mIgM-containing BCRs in the plasma membrane of human B cells. a Schematic depiction of the staining procedure and the generation of plasma membrane sheets. b, e, h Images of membrane sheets of unstimulated Ramos B cells (b), unstimulated primary B cells (e), and Ramos cells stimulated with anti-IgM F(ab’)2 fragments for 30 min on ice (h). Membrane sheets were stained with the membrane marker R18 (confocal images pseudo-colored in green) and anti-human IgM affibody-Star635P (STED images displayed in red). White squares on central images indicate the zoomed regions that are displayed in the images to their right. Scale bars represent 5 µm and 1 µm for the low and high zoom images respectively. c, f, i Example histograms showing the fluorescence intensity distributions of single Star635P-conjugated affibodies on coverslips (black curves, control) and mIgM-spots present in membrane sheets (red curves, mIgM-BCRs). d, g, j Percentages of different mIgM-BCR arrangements in plasma membrane sheets were calculated after pooling the spot intensities of dozens of membrane sheets together and then fitting the intensity distribution to a sum of Gaussian curves (Supplementary Fig. 2). Data were obtained from four and three independent experiments including a total of 30 membranes from unstimulated and 15 membranes from F(ab’)2-stimulated Ramos B cells, respectively. The experiments with primary human B cells were performed three independent times from two different donors and 30 membrane sheets were analyzed in total. Error bars represent the 68% confidence interval, corresponding to 1 standard deviation. Histogram’s source data are provided as a Source Data file