Ti2eclipse inverted microscope

For epifluorescence imaging, all Z-stack images were obtained with 0.5 microns between slices. Images were acquired in an epifluorescence microscope (Nikon Ti2Eclipse) with an X60/1.25NA and X100/1.3NA oil immersion objectives for bead and spreading assays, respectively. For confocal microscopy, images were acquired in a Nikon Ti2Eclipse inverted microscope with 60X/1.45NA oil immersion for bead and spreading assays, with a Z-stack of 0.5 microns. For Total internal reflection fluorescence microscopy (TIRFM), images were acquired in Nikon Ti2Eclipse inverted microscope with a 100x/1.50 NA oil immersion lens and an iXON Ultra EMCCD camera at 37°C. B-cells expressing LifeAct-mCherry were plated on Ag-coated glass chambers (Nunc^TM Lab-Tek^TM II). Images were acquired for 30 min at 15 s per frame for spreading assay and for 1 min at 0.75 s per frame for lysosome, proteasome, and actin retrograde flow tracking. For Ayriscan acquisition, images were obtained in the Zeiss LSM880 Airyscan Confocal microscope with a 63X/1.4NA oil immersion lens, with a Z-stack of 0.2 μm. The images were processed using Zeiss Black Zen software and analyzed with FIJI.

Images were acquired on a Nikon Ti2 Eclipse inverted microscope using a Plan Apo Lambda DM 60× (1.4 NA, Ph3) oil objective and an Andor Zyla sCMOS camera. Images were acquired using the NIS-Element AR software.

Total internal reflection fluorescence microscopy (TIRFM) images were acquired in Nikon Ti2Eclipse inverted microscope with a 100x/1.50 NA oil immersion lens and a iXON Ultra EMCCD camera at 37°C. B-cells expressing LifeAct-mCherry were plated on Ag-coated glass chambers (Nunc™ Lab-Tek™ II). Images were acquired for 30 min at 15 s per frame for spreading.