Super-Resolution Imaging of Cell Interactions

STORM analysis was performed as previously described (47 (link)). In brief, WBC were isolated from 4T1-tumor bearing mice and incubated in 5 ml glycine quenching buffer for 30 min at RT. The cells were incubated with the LQI tetramer SA-Alexa 568 and CD177-Alexa 647 antibody for 30 min at 4°C. Following washing and fixation (PFA) the cells were stained with YOYO-1. Before imaging, cells were resuspended in STORM imaging buffer (100mM MEA, 10% glucose, Glox (11.2 mg/ml glucose oxidase (Sigma) and 1.8 mg/ml catalase (Sigma)) in dilution buffer (50 mM NaCl, 200 mM Tris in D₂O)). STORM was performed in a Nikon Eclipse Ti-E microscope with a CFI Apo TIRF × 100 DIC N2 oil objective (NA 1.49, WD 0.12 mm) equipped with a Andor iXon-897 EMCCD camera. Super-resolution images were reconstructed from a series of at least 5000 images per channel using the N-STORM analysis module, version 1.1.21 of NIS Elements AR v. 4.40 (Laboratory imaging s.r.o.). Co-localization analysis was performed using the ImageJ plugin ‘Interaction Factor package’ described previously (35 (link)). The calculated ‘Interaction Factor’ scores between 0 and 1, where 0 represents no interaction and 1 represents complete overlap.