B cell spreading in response to substrate stiffness depends on BCR activation and is unaffected by stiffness alone. (A) Illustration of basic workflow used to activate B cells on PAA gels. Cells are seeded for different time points and then fixed, used for live imaging, or lysed for protein extraction. (B) AFM measurements in kPA of gel stiffness. Soft gels have values of 0.3 kPa, and stiff, 13 kPa. (C) Top: Representative confocal image for the Z cross section of a PAA gel coupled to a fluorescently labeled BCR⁺ ligand. Scale bar: 2 μm. Bottom: Analysis for antigen distribution (BCR⁺ ligand) based on MFI values acquired in confocal microscopy. (D) Representative images of B cells seeded on soft and stiff gels coupled to a BCR⁻ ligand (nonactivating). Cells were labeled with phalloidin (red) and do not display spreading responses. Scale bar: 5 μm. (E) Quantification of the spreading area from experiments depicted in D. Shown data consider n ≥ 30 cells pooled from N = 3, independent experiments. (E): Two-way ANOVA with Sidak’s multiple comparison test. (B and C): t test. P values illustrated with asterisks are * <0.05 and *** <0.001.