Ix71 inverted epi fluorescent microscope

Cells were stained with DiO prior to the experiment. 2,000 cells were resuspended in 1μL of ECM mixture and plated on one end of a well on a 4 well Labtek plate (Thermo Scientific, cat#154917). 0.8ng of CXCL9 (R&D systems, cat# 392-MG) was resuspended in 2μL of ECM and plated on the other end of a well. The ECM mixture consisted of 20% growth factor reduced Matrigel (Corning, 10–12 mg/ml stock concentration, #354230) and 80% rat tail collagen type I at 3mg/mL (Gibco, A1048301). The two droplets were then covered in 75μL of ECM and was allowed to solidify for 45 minutes at 37°C. 800μL of NK media was then added to the well and the cells were allowed to migrate for 24 hours. The entire slide was then imaged on the Olympus IX-71 Inverted Epifluorescent Microscope at 5X in the LCCC Microscopy Shared Resource and the number of cells that had migrated beyond the initial droplet were counted using FIJI.

Myc-BirA^∗-Txnip was generated by first generating Myc-BirA^∗ by PCR from the pcDNA3.1 mycBioID plasmid [20 (link)]. PCR products were digested and ligated into pIRES2-EGFP (Clontech). Next, pCR4-TOPO-Txnip (Thermo Fisher Scientific) was used as template to amplify Txnip which was ligated in frame with Myc-BirA^∗ in pIRES2-EGFP. Subsequently, Myc-BirA^∗-Txnip^C247S was generated by site-directed mutagenesis (Agilent Technologies). Plasmids were linearized and transfected into HEK293 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were selected in 200 μg/mL hygromycin and stable clones were selected based on EGFP fluorescence visualized with an Olympus IX71 inverted epifluorescent microscope.