Flexmap 3d machine

Gastric tissues were lysed and processed using a Mouse Milliplex kit (Millipore) and analysed on a Luminex FlexMap 3D machine[17 (link)]. CXCL1 was also measured using the DuoSet ELISA Mouse CXCL1 kit (R&D Systems).

For barrier function analysis, mice were gavaged orally with 150 μl of 80 mg/ml fluoroscein isothiocyanate (FITC) dextran (Sigma) dissolved in sterile PBS (6d after tmx injection). Four hours after gavage, mice were sacrificed, and blood was collected by cardiac puncture. Serum was isolated by centrifugation, diluted 1:10 in PBS and loaded onto a black, opaque bottomed microplate for measurement using a Synergy HT plate reader (BioTek) excited at 485 nm and read at 530 nm. Concentration was determined using a FITC-dextran standard curve. To determine colony formation, whole blood samples were diluted 1:2 or 1:10 in sterile PBS and 100 μl was plated on blood agar plates (Fisher). Plates were incubated at 37°C for 20 hours and colonies were quantified. For cytokine analysis, serum was analyzed by MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Immunology Multiplex Assay (Millipore) using a Luminex Flexmap 3D machine in conjunction with the UPenn Human Immunology Core.