(A, B) Statistical quantification of the synaptic accumulation of IgG-BCR and the volume of the contact area of J558L cells expressing memory B1-8-IgG-BCR encountering 12 pN, 43 pN, or 56 pN NP-TGT sensors. (C, D) Statistical analyses of synaptic accumulation of pSyk accumulation (C) and the volume of pSyk microcluster (D) in response to 12 pN, 43 pN, or 56 pN NP-TGT sensors. (E, F) Quantification of the synaptic accumulation of IgG-BCRs (E) or pSyk (F) in memory B cells expressing isotype-switched B1-8-IgG-BCR from B1-8 Tg mice that were placed on coverslip presenting 12 pN, 43 pN, or 56 pN NP-TGT probes. (G) Representative TIRFM images showing the dynamics of the synaptic accumulation of IgG-BCRs from J558L cells expressing memory B1-8-IgG-BCR in contact with coverslip presenting 12 pN, 56 pN NP-TGT sensor, or control TGT (NC) molecule at the indicated time points. Scale bar is 1.5 μm. (H) Comparisons of averaged traces showing the dynamic accumulation of memory IgG-BCRs as demonstrated in (G) in a 13 min TIRFM imaging time course. Bars represent mean ±SEM. Data were from at least 20 cells over three independent experiments. (I–L) Statistical analyses of the synaptic accumulation of two types of chimeric BCRs and pSyk, Human mIgE heavy chain (I, J), or Human mIgM heavy chain (K, L) with mouse B1-8 variable region in human Ramos B cells encountering 12 pN, 43 pN, or 56 pN NP-TGT sensors. In all of these plots, bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data were from at least 30 cells in each group of three independent experiments.