Cat no 3464

Concentration gradients of VEGF in transwell assays and in the gLL were modeled in COMSOL Multiphysics 5.3a (COMSOL Multiphysics 5.3a, COMSOL Inc., Burlington, USA) using physical properties of VEGF and the true geometry of the assays, as per manufacturer references and our own measurements (Cat No. 3464, Corning Incorporated, NY). Transport of EGF and VEGF within the gLL and transwell assay was computationally modeled to predict reagent concentration over time across the microchannel and permeable membrane, respectively. The diffusivity of EGF was estimated to be 2.0 × 10⁻⁶ cm²/s for EGF [45 (link)], and 9.0 × 10⁻⁷ cm²/s for VEGF using the Stokes–Einstein equation [46 (link),47 (link),48 (link)].

Boyden chambers (VWR, PA) in combination with thin porous membranes were used as transwell assays for this study. rMC-1 were seeded at a concentration of 2.5 × 10⁵ cells/mL in serum-free DMEM on the top part of a two compartment transwell model, divided by an 8 µm porous polyester membrane of 10µm thickness (Cat No. 3464, Corning Inc., ME), as per Figure 2. The bottom compartment was filled with either EGF or VEGF at 100ng/mL in DMEM for test conditions or serum-free DMEM for the control group. rMC-1 were allowed to migrate for 6 h at 37 °C and 5% CO₂. Upon completion, the top surface of the membrane was cleaned with an applicator followed by fluorescent staining of the bottom surface of the membrane, containing the cells that migrated through it, using Cell TrackerTM Green CMFDA (Cat No. C7025, ThermoFisher, MA) at a concentration of 15 µM. Numbers of motile rMC-1 located at the bottom side of the porous membrane were optically measured for all tests. rMC-1 migration in response to ligand signaling through these transwell assays was examined using four conditions: (i) rMC-1 stimulated by EGF signaling fields; (ii) rMC-1 stimulated by VEGF signaling fields; (iii) rMC-1 exposed to VEGF for one hour and then stimulated by EGF signaling; and (iv) control (i.e., media only, no gradients). Each testing condition was examined in triplicate.