Fig 2: Mechanosensitive cation channels exclusively mediate nanopore‐mediated B cell activation but not activation via CD40 and IgM crosslinking. B cell morphology based on phalloidin staining (yellow) with additional pBTK (magenta) and IgM immunolabeling (green) following incubation with GsMTx‐4 (A) and GsMTx‐4/Thapsigargin (B) for 1 hr and subsequent plating on substrates for 30 min prior to fixation, IHC, and microscopy. Bottom panels in (A, B) show a sideways view (xz) of boxed ROIs in upper panels (xy), with a turquoise dotted line demarcating the surface of nanoporous substrates. (C) Microvilli numbers per cell. (D) Average length of phalloidin, pBTK, and IgM signals within phalloidin+ve microvilli. (E) pBTK intensity quantification within phalloidin+ve microvilli. Cell surface expression of the activation marker CD69 on B cells (CD69+ve, CD19+ve) stated as % of parent live B cell population following 24 h incubation on flat, flat (αCD40, αIgM), and nanopore substrates in the presence of GsMTx‐4 (F), GsMTx‐4/Thapsigargin (G), GsMTx‐4/Cytochalasin D (H). Scale bars, 5 µm (A, B) unless stated otherwise. N = 3 biological repeats (independent blood donors), n = total 57 cells (GsMTx4), 55 (GsMTx‐4/Thapsigargin). Data show mean ± SD. One‐way ANOVA (C‐H); n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 3: The activation marker CD69 is upregulated on B cells after 24 h on nanoporous substrate. (A) Representative flow cytometry gating strategy for cell surface marker analysis at 24 h on nanoporous substrate. (B) Viability analysis of gated lymphocytes stated as % ZombieNIR−ve of parent single lymphocyte population. (C) Purity assessment of B cells (CD19+ve) as opposed to T cells (CD3+ve) within cell cultures stated as % of parent live lymphocyte population (see also A). (D) Cell surface expression of the activation marker CD69 on B cells (CD69+ve, CD19+ve) stated as % of parent live B cell population following 1 day of in vitro incubation. (E) Cell surface expression of the activation marker CD69 on B cells (CD69+ve, CD19+ve) stated as % of parent live B cell population following 5 days of in vitro incubation. (F) Cell surface expression of the activation marker CD80 on B cells (CD80+ve, CD19+ve) stated as % of parent live B cell population following one and 5 days of in vitro incubation. (G) Cell surface expression of the activation marker CD83 on B cells (CD83+ve, CD19+ve) stated as % of parent live B cell population following one and 5 days of in vitro incubation. N = 10 biological repeats (independent blood donors) for B–D. N = 3 biological repeats for E‐G. One‐way ANOVA (B‐E). Data show mean ± SD. Two‐way ANOVA (F, G); ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 4: B cell activation on nanopores is not exclusively mediated by pBTK, general cytoplasmic Ca2+ signaling, or actin cytoskeleton dynamic behavior. Cell surface expression of the activation marker CD69 on B cells (CD69+ve, CD19+ve) stated as % of parent live B cell population following incubation on flat, flat (αCD40, αIgM), and nanopore substrates in presence of Ibrutinib (A), BAPTA‐AM (B), Thapsigargin (C), Ionomycin (D), Cytochalasin D (E), and Jasplakinolide (F). N = 3 biological repeats (independent blood donors). Data show mean ± SD. One‐way ANOVA (A–E); n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. See also Figures S4A–P and S5A,B.