Endotoxin quant kit

The cell culture medium LHC-9, DMEM:F12 and Trypsin–EDTA were bought from Gibco, Thermo Fisher Scientific, Waltham, MA, USA. The cell culture flasks were obtained from Nunc A/S, Roskilde, Denmark, while the cell culture plates were from Corning, NY 14831 USA. PureCol collagen was from Advanced BioMatrix, Inc, CA, USA. The Cytotoxicity Detection Kit using lactate dehydrogenase (LDH) activity was bought from Merck KGaA, Darmstadt, Germany. AlamarBlue™ Cell Viability Reagent and the sandwich enzyme-linked immunosorbent assay (ELISA) cytosets for CXCL8 and IL-6 were purchased from Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, while IL-1α and IL-1β DuoSet were from R&D Systems, Inc, Minneapolis, MN, USA. The Endotoxin Quant kit were from Thermo Fisher Scientific, Waltham, MA USA. For measurements of acellular ROS 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), Dulbecco’s PBS; hydrogen peroxide (H₂O₂), dithiothreitol (DTT) and ultrapure H₂O were purchased from Merck KGaA, Darmstadt, Germany. Whatman TE 38 filter was from (Whatman, Maidstone, Kent, UK). Other chemicals were purchased from commercial sources at the highest purity available.

Thirty mice were randomly divided into five groups: negative control (NC), positive control (PC), IL10-NP, IL10-AMNP, and Free IL10. High-endotoxin HDM (Greer, USA; lot: 378908, 192250 EU per vial or 4970 EU/mg) was removed using an endotoxin removal standard spin column (Cat#PUR028, AbD Serotec, UK). After through the column, the concentration of endotoxin was 1569 EU/mg, which was quantified using an Endotoxin Quant Kit (Cat #A39553, Thermo Fisher, USA). Mice were anesthetized with isoflurane and sensitized on days 0, 2, and 4 with 25 µg HDM or PBS intranasally. Challenges were performed with 25 µg HDM in 25 µl PBS from days 14 to 18. The treatment groups received 0.1 µg IL-10 simultaneously with the HDM challenge. The NC group received PBS at all time points.