The dual hydrogel culture system was fabricated on the membranes of Transwell ® 6-well inserts (0.4 µm/PES; Corning) using digital projection photolithography as previously described (Curley and Moore, 2011; (link)Bowser and Moore, 2019) (link). The outer cell-impermeable hydrogel was created using a photo-translinkable solution of 10% w/v polyethylene glycol dimethacrylate 1000 (PEGDMA; Polysciences, Warrington, PA) and 0.55 mM lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Sigma-Aldrich, St. Louis, MO). The photo-translinkable solution was irradiated for 25-30 sec using ultraviolet light of 385 nm wavelength at 300 W/m 2 with a digital micromirror device (DMD) light engine (PRO4500 Wintech Digital Systems Technology Corp, Carlsbad, CA). A mask design and polymerization parameters were selected using commercial software (DLP Lightcrafter 4500 Control Software, Texas Instruments, Dallas, TX). The mask shape resembles a keyhole, where the circular reservoir is referred to as the "bulb" and the narrow slot is the "channel". The constructs were washed using 2% antibiotic/antimycotic wash buffer (Thermo Fischer Scientific, Walton, MA) and the channels were filled with 8% Growth Factor-Reduced Matrigel ® Matrix (Corning, Corning, NY) to create a cell-permeable scaffold.
Transwell 6 well inserts
Manufactured by Corning
Sourced in United States
Transwell® 6-well inserts are a component of a cell culture system designed for in vitro studies. They consist of a porous membrane suspended within a well, allowing for the creation of a barrier between two compartments. This design facilitates the study of cell-cell interactions, permeability, and transport processes.
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Lab products found in correlation
2 protocols using transwell 6 well inserts
1
Dual Hydrogel Scaffold Fabrication
2
Chemotaxis Assay for THP-1 Cells
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For TNF‐α stimulations, THP‐1 cells were starved in 1%FBS RPMI 1640 medium (with pen/strep) overnight. AVE0991 were added to THP‐1 cell culture 18 h before TNF‐α (10 ng·cm−3), 6 h stimulation. THP‐1 cells were centrifuged for 5 min at 400 x g and suspended in DMEM 1%FBS with pen/strep and 1 × 106 cells were added to 8 μm pore Transwell® 6 well Inserts (Corning®, US). Supernatants were added to the bottom part and left for 4 h at 37°C in a 5% CO2. Cells from bottom part were collected and counted by flow cytometry (LSRII, BD). Fold change of chemotactic properties were calculated comparing cell number passing through the pores to the THP‐1 cells stimulated with TNF‐α and passing through to supernatant from SW872 cells, without stimulation.
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