Figure 1
The SHIP assay can be used to track antigen internalization in B cells. (A) A20 D1.3 B cells imaged with a SDCM. Cells were stained on ice with AF488 anti-IgM to visualize the surface-bound BCR, fixed and stained with DAPI (nucleus). (B) Schematic of the SHIP assay. B cells are incubated with FIP-IgM as a surrogate antigen to label and activate the BCRs. B cell activation will trigger antigen/BCR internalization. After QP addition, the surface-bound probes are quenched and only the signal from the internalized antigen will be detected. (C,D) B cells were stained with ATTO-FIP-IgM and anti-IgM Alexa Fluor 488, and activated at different time points. After activation, cells were incubated with (C) PBS or (D) QP for 10 min and fixed. Images were acquired using a SDCM. A representative image of one cell (stack, sum intensity projection) is shown. White and red circles mark examples of internalised and non-internalised antigen, respectively. Scale bar: 5 µm. (E) Colocalisation analysis of the images shown in C-D. Images were deconvoluted with Huygens and colocalisation was measured using Mander’s overlap coefficients (tM1 = % of the anti-IgM Alexa Fluor 488 signal colocalising with ATTO-FIP-IgM signal; tM2 = % of the ATTO-FIP-IgM signal colocalising with anti-IgM Alexa Fluor 488 signal). Different colours represent independent experiments (n = 3): the mean of each experiment is shown with large symbols, and small symbols show the measured cells. Statistics were calculated using the mean of each experiment (n = 3) and a paired t-test (*P < 0.05; #P < 0.05 vs 10 min + QP; ###P < 0.001 vs 5 min + QP).
