No 1.5 german borosilicate coverglass

A quantity of 100 μl of growth factor‐reduced Matrigel (GFR‐MG) (8 mg/mL; 2:1 GFR‐MG: DMEM‐Ham's F12 [1:1] medium; Becton Dickinson, Franklin Lakes, NJ) was allowed to solidify in a 37°C incubator for 1 h in eight‐well chambers mounted on No. 1.5 German borosilicate coverglass (Nalge Nunc International, Naperville, IL). Then, Par‐C10 cells (15,000 cells/well; passages 30–60) were plated on the GFR‐MG in DMEM‐Ham's F12 (1:1) medium with supplements and treated with TNF‐α (100 ng/mL; Becton Dickinson), then incubated for 1 h or 6 h at 37°C. Cells were then treated with AT‐RvD1 (100 ng/mL; Cayman Chemical, Ann‐Arbor, MI), and varying doses of DEX (25–100 ng/mL). Three‐dimensional Par‐C10 cell clusters were used for assays after incubation at 37°C with 95% air and 5% CO₂ for 72 h.

One hundred microliters of matrigel (BD Biosciences) (2:1 matrigel:media) was allowed to solidify in a 37°C incubator for 1 hour in 8-well chambers mounted on No. 1.5 German borosilicate coverglass (Nalge Nunc International, Naperville, IL). Upon solidification, 3,000 cells/well were seeded on the matrigel in media, as indicated. 3D-spheroids were grown at 37°C in 5% CO₂. To generate single cell suspensions from 3D-spheroids, spheres were isolated by removing media and adding 0.5ml of Dispase (1mg/ml) directly to the matrigel followed by incubation for 1 hour at 37°C in 5% CO₂ to digest the matrigel. Spheres were washed in PBS and centrifuged at 1200rpm for 5min. Spheres were dissociated with TrypLE Express (Gibco) and passed through a 40μm filter to remove clumps and obtain single cell suspensions. Cells were then plated and grown as monolayers in either SFM or SCM.