TIRF imaging data of neutrophils migrating underneath endothelial cells

This data is used in the publication "Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling": https://www.frontiersin.org/articles/10.3389/fimmu.2021.667213/full

TIRF Microscopy

Lentiviral transduction was used to generate an endothelial cell line expressing mNeonGreen-Paxillin (derived from addgene plasmid # 129604). Cells were imaged with a Nikon Ti-E microscope equipped with a motorized TIRF Illuminator unit, a 60x TIRF objective (60x Plan Apo, Oil DIC N2, NA =1.49, WD = 120 um) and Perfect Focus System. Images were acquired with an Andor iXon 897 EMCCD camera and the Nikon NIS elements software. mNeonGreen was imaged using the 488 nm laser line and calcein red-orange was imaged using the 561 nm laser line. A quad split dichroic mirror (405 nm, 488 nm, 561 nm, 640 nm) was used in combination with dual band pass emission filter (515 to 545 nm, 600 to 650 nm). To achieve a larger field of view a 3 x 3 tile scans was acquired with 15% overlap stitching on the GFP channel. Time lapse images were taken every 10 s.