Single-cell force cytometry using Fluorescently Labelled Elastomeric Contractible Surface (FLECS) technology B

BSMCs were seeded on Fibronectin-coated elastomeric micropatterns (Forcyte Biotechnologies #F2AX0G03Y, 50 microns, Alexa Fluor 488 - bound Fibrinogen, 24-well plate) at a concentration of 75,000 cells per well in Smooth muscle growth medium (SmGM) (Lonza) and left to adhere and spread for 90 minutes. Unattached cells were then removed and fresh SmGM medium supplemented with rhTNF-α (10 ng/mL)/rhIL-13 (100 ng/mL)/ACh (10μM) was added. BSMCs were stimulated for 3 hrs before being fixed in pre-warmed to 37°C 4% PFA. Fixed samples were washed and then stained with ATTO-565 phalloidin (ATTO-TEC) and DAPI (Life Technologies) and imaged on a StellarVision microscope using Synthetic Aperture Optics technology (Optical Biosystems). All images were analyzed using FiJi software by measuring the length of each micropattern per condition after stimulation and subtracting the length of the unstimulated micropattern of the same condition.