A MFP-3D-Bio AFM (Asylum Research, Santa Barbara) was used for all AFM measurements. Changes were made to our previously described protocol,24 (link) to optimize measurements of the ELS in mouse ECs. For all AFM measurements, each force curve indented a distance of 250 nm after contact at a rate of 4 μm/s. Separate force curves on a 32 × 32 grid spanning 90 × 90 μm over several cells were collected to a 200pN trigger point, and the Hertz model was applied to the first 100 nm of indentation through the Asylum Research software. Given that all three cell types were similar in height, a single AFM protocol was used. Curves were manually inspected for goodness of fit and contact point was adjusted as needed. Bad fits were discarded from the analysis. Stiffness measurements were taken with a TR400PB, silicon nitride tip, with a height of 3 μm and a semi-included angle of 35° (k = 0.02 N/m). The comparison of live and fixed ECs was performed on BD Falcon Petri dishes (BD Biosciences, San Jose, CA).