AFM-FS Measurements with Cytochalasin D and IgM

For treatment for AFM-FS measurement, Cytochalasin D (Sigma, C8273, Albuquerque, New Mexico) was added directly to PBS, incubated for 10 minutes, and then maintained in the plate during the measurement. Similar protocol was used for treatment with IgM (Southern biotech, 9023-01, Birmingham); 10 µL/mL of IgM was added directly to PBS and maintained in the medium during all measurements. The experiment lasted no longer than 2 hours to avoid saturation of the BCR signal. Ibrutinib (Selleckchem, S2680, Planegg, Germany) and acalabrutinib (Selleckchem, S8116, Planegg, Germany) were administered at concentrations of 1 μM or 10 μM^{40 (link)} to cells in suspension. After 4 hours incubation, the unbound drugs were washed out by centrifuging the cell suspension. Cells were then seeded on polyornithine precoated dishes (1:10) for 2 hours to obtain stable adhesion, as described above for AFM-FS experiments in the absence of the drug.