Recombinant rat tnf α protein

The MSCs were seeded in 12-well plates at a density of 1 × 10⁵MSCs/well with or without embedding in 600 µl of 4 mg/ml dECM solution. The dECM
solution-MSC mixture suspended in plates was incubated at 37°C for 30 min for
gelation. After confirming gelation, 500 µl of MesenPRO RS™Medium (Thermo Fisher
Scientific K.K.) with Zell shield (Minerva Biolabs, Inc.) was added to each well
and incubated for 24 h. The culture medium was then replaced with 500 µl of
culture medium with or without 10 ng/ml of recombinant rat TNFα protein (R&D
Systems, Inc., MN, USA). Following 48 h of cell culture, TSG-6, and HGF secreted
in culture supernatants were measured by enzyme-linked immunosorbent assay
(ELISA), using rat tumor necrosis factor-inducible gene 6 protein (TSG-6) ELISA
kit (WUHAN HUAMEI BIOTECH Co., Ltd, Wuhan, China) and rat HGF Quantikine ELISA
kit (R&D Systems, Inc., Minneapolis., MN, USA) according to the
manufacturer’s instructions. The absorbance was measured by GloMax® Discover
(Promega Co.). Normalization was performed with the estimated number of live
cells calculated based on the absorbance values from the live/dead staining
assay. Cultured cells were collected for quantitative Real-Time PCR
analysis.