Figure 2

Colocalisation of the antigen with early and late endosomal markers. (A,B) B cells were engaged with ATTO-FIP-IgM and anti-IgM AF488, and activated for 10 or 30 min at 37 °C. After activation, cells were incubated with QP for 10 min on ice, fixed and stained for (A) Rab7 or (B) EEA1. Images were acquired using a SDCM. A representative image of one cell (stack, sum projection) is shown. The white and yellow circles mark an example of internalised or non-internalised antigen, respectively. Scale bar: 5 µm. (C) Colocalisation analysis of the internalised antigen with EEA1 and Rab7. B cells were stained with ATTO-FIP-IgM and activated for 10 or 30 min. After activation, cells were incubated with PBS (−QP) or QP (+QP), fixed and stained for EEA1 and Rab7. Images were deconvoluted with Huygens and colocalisation was measured using thresholded Mander’s overlap coefficient (tM1 = % of antigen colocalising with EEA1 or Rab7). Different colours represent independent experiments: the mean of each experiments is shown with large symbols, and small symbols show the measured cells. As a control for random colocalisation, one channel was rotated 90° and colocalisation was measured. **P < 0.01, paired t-test (n = 3).