Chambered coverglass slides (Lab-Tek II, Nunc) were coated with 0.01% poly-l-lysine (PLL) (Sigma) for 20 min at room temperature before being washed in water and dried for 1 hour at 60°C. Under unstimulating conditions, cells were allowed to adhere to PLL for 20 min at 37°C. The mAbs were coated at various concentrations in PBS onto PLL-coated coverglass slides overnight at 4°C. After washing and blocking the slides in culture medium, the cells were allowed to interact with the coated surfaces for 5 min at 37°C. Cells were fixed in 4% PFA/PBS (Thermo Fisher Scientific) for 30 min at room temperature and then washed in PBS. Samples for PALM analysis were imaged immediately, whereas samples for TIRF, STORM, and STED analyses were either stained immediately or stored overnight at 4°C with 0.02% azide/PBS. For surface protein imaging, after cells were fixed, they were incubated in blocking buffer 1 (1% BSA and 1% human serum/PBS) for 1 hour at room temperature, then stained in blocking buffer 1 containing antibodies against surface proteins at appropriate concentrations (listed earlier), as determined by titration for 1 hour at room temperature, and then washed in PBS. For assessment of their spreading responses, cells were fixed, permeabilized, and blocked with 0.1% saponin (Sigma) in blocking buffer 1 for 1 hour. Saponin-mediated cell permeabilization is reversible, and so, saponin was maintained in the washing and blocking steps thereafter. Cells were stained with phalloidin–Alexa Fluor 568 or phalloidin–Alexa Fluor 488 (Life Technologies) at 13.2 nM (1:500 dilution), directly conjugated antibodies for confocal imaging for 1 hour at room temperature, and then washed in PBS. In experiments in which cells were stained with phalloidin alone, the cells were permeabilized with 0.1% Triton X-100/PBS (Sigma) for 5 min at room temperature. For signaling protein imaging, after cells were fixed, they were incubated with lysophosphatidylcholine (100 μg/ml)/PBS (Sigma) for 10 min at room temperature [a reversible permeabilizing agent used previously for membrane proteins (50)]. Cells were washed and then incubated with blocking buffer 2 [1% goat serum and 4% BSA/50 mM tris-buffered saline (TBS)] for 1 hour at room temperature, followed by incubation with blocking buffer 2 containing primary antibodies for 1 hour at room temperature. After washing, the cells were incubated with goat secondary antibodies conjugated to Alexa Fluor 568 (Thermo Fisher Scientific) in blocking buffer 2 for 1 hour at room temperature and then washed in TBS. After staining, cells were postfixed with 4% PFA/PBS for 5 min at room temperature and then washed. Samples for STORM and TIRF analyses were either imaged immediately or stored overnight at 4°C in 0.02% azide/PBS. Samples for STED analysis were mounted in a hard-drying medium that preserved fluorescence and matched the refractive index of the STED microscope lens (Fluorescent Mounting Medium, Dako).

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