Ensight multimode reader

PGE₂ ELISA was performed with fresh culture supernatants from MSC-macrophage co-cultures using a PGE₂ forward sequential competitive ELISA kit (R&D Systems; USA) as per manufacturer’s instructions. Absorbance was read at 450 nm with a wavelength correction of 540 nm on an EnSight multimode reader (Perkin Elmer).

HEK293 cells were transfected with Peroxisome Proliferator Response Element (PPRE)-driven Firefly luciferase (addgene 1015) and SV40-driven Renilla luciferase coding vectors together with PPARγ or PPARα expression vector. PPARγ-LBD-Gal4 or PPARα-LBD-Gal4 expression vector (given by Dr. Teruo Kawada, Kyoto University, Japan) was transfected along with the SV40-driven Renilla luciferase expression vector in HEK293 cells stably expressing the Gal4 response element-driven firefly luciferase reporter (pGL4.35(luc2P/9XGAL4UAS/Hygro) vector from Promega, Madison, WI, USA). Thirty-six hours after transfection, cells were exposed to the tested compounds for an additional 16 h; then, firefly and Renilla luciferase activities were measured in the cell lysates using the reagents Genofax A and C (Yelen) in an EnSight multimode reader (Perkin Elmer, Courtaboeuf, France). PPAR transactivation activity of the compounds is calculated as ratio of firefly to Renilla luciferase activity.

The effect of the HA nonwoven textiles on immune cell activation was assessed using the Monocyte Activation Test (MAT). The textile samples were dissolved in normal saline (0.9% NaCl) at a concentration of 1 mg/mL. Heparinised whole blood from four healthy donors was pooled and 10× diluted with normal saline. The diluted blood was placed in sterile microtubes and 100 µL of the sample solution was added, followed by incubation at 37 °C for 16 to 20 h. In parallel, the inner control sample was prepared by further adding Reference Standard Endotoxin (RSE, Merck, Darmstadt, Germany, cat. no. #E0150000) at a concentration of 0.25 EU/mL to the sample solution. The purpose of the inner control sample was to exclude undesired interaction between the sample and the RSE lipopolysaccharide. For evaluation purposes, also solutions of pure RSE at concentrations of 10, 5, 1, 0.25 and 0.1 EU/mL were tested.
After incubation, the microtubes were manually shaken and centrifuged at 13 000 rpm for 10 min. The upper phase (plasma diluted in the saline) was collected for IL-6 analysis using an IL-6 Human Uncoated ELISA kit (Invitrogen, Waltham, MA, USA, #88-7066-88) according to the manufacturer’s protocol. Absorbance was read by an EnSight Multimode Reader and the Kaleido software (both from PerkinElmer, Waltham, MA, USA) was used for evaluation.