Single Particle Tracking of Receptor Dynamics

Daudi B cells were washed once in PBS then stained in 2% FBS/PBS at a concentration of 1 × 10⁶ cells/ml with 4 ng/ml of Attotec® 488 labeled goat anti-human IgM Fab fragment and 1 μg/ml of Attotec® 633 labeled pinatuzumab Fab fragment for 20 min at 4°C. Cells were washed two times with PBS and resuspended in chamber buffer (0.5% FBS, 2 mM MgCl₂, 0.5 mM CaCl₂, and 1 g/L D -glucose in PBS). Cells were incubated at 37°C for 5 min before injecting in FSC2 chambers preheated to 37°C. Single particle tracking (SPT) was performed on a total internal reflection fluorescence (TIRF) microscope (Quorum Technologies) based on an inverted microscope (DMI6000C, Leica), HCX PL APO 100X/1.47 oil immersion objective and Evolve Delta EMCCD camera (Photometrics). Cells were allowed to settle in chambers for 1 min prior to image acquisition and images were taken up to 10 min. Images were acquired continuously at 20 frames/s for 10 s with an EM Gain of 200 and an exposure time of 50 ms. SPT analysis was performed on a custom MATLAB script as described previously (25 (link)).