Elisa

BAL supernatant was separated into two fractions using differential centrifugation. 1 mL of BAL from the first wash was centrifuged at 18,000 rpm for 30 min at 4 °C to obtain large and small aggregate fractions. The large aggregate (LA) fraction (pellet) contains phospholipids, tubular myelin, lamellar bodies, large vesicles, SP-A, B and C. The supernatant contains the small aggregate fraction (SA) consisting of small vesicles, SP-D and little surface functional surfactant [27 (link)]. The LA was re-suspended in 40 μL of physiological saline and 5 μL was separated into organic and aqueous fractions by a chloroform and methanol extraction. The lower layer containing the organic fraction was dried under nitrogen. Total organic phosphorus was extracted using a perchloric acid digestion (70 %) for one hour at 200 °C, with potassium phosphate standards treated in the same way as the samples and assayed using the method of Bartlett [28 (link)]. Organic phospholipid was expressed as μg of total organic phosphate in 5 μL LA, equivalent to 1/5 mL of BAL. Protein concentrations in the LA and SA were determined by the Bio-rad assay. SP-D was measured in whole BAL supernatants by ELISA [Cusabio Biotech Co., Suffolk, UK] and SP-B inthe LA fraction by ELISA [Cusabio Biotech Co., Newmarket, Suffolk, UK.

Individual culture cell-free supernatants were collected and clarified by centrifugation. The concentration of human soluble P2X7 was tested by ELISA following the manufacturer’s instructions (Cusabio, Houston, TX, USA). Plasma levels of human ASC, HMGB1, GSDMD and P2X7 were also tested by ELISA (Cusabio for ASC and P2X7, Aviva Systems Biology (San Diego, CA, USA) for GSDMD and Arigo Biolaboratories (Hsinchu, Taiwan) for HMGB1). Results were read in a Synergy Mx (BioTek, Winooski, VT, USA) plate reader at 450 nm and corrected at 540 nm.

All animals underwent an OGTT at 4 weeks after the implant of the osmotic pump. After an overnight fasting, the rats received a 50% D-glucose solution (1 g/kg body weight) by oral gavage. Blood samples were taken by tail bleeding and collected in EDTA tubes. All blood samples were immediately centrifuged and plasma divided into appropriate subsamples and stored at −20 °C for further analysis. Blood glucose, plasma insulin and C-peptide were measured at 0, 20, 40, 60, 80, 100, 120, and 180 min. Blood glucose levels were measured by glucometer (Accu-Chek, Roche Diagnostics Division, Grenzacherstrasse, CH). Plasma insulin was measured by ELISA (EMD Millipore Corporation, Billerica, MA), with a sensitivity of 0.1 ng/ml and an intra- and interassay precision of 1.9% and 7.6%, respectively. C-Peptide was measured by ELISA (RayBiotech, Peachtree Corners, GA) with a sensitivity of 772 pg/ml and an intra- and interassay precision <10% and <15%, respectively. C-reactive protein (CRP) was measured by ELISA (Cusabio, Houston, TX) with a sensitivity of 7.81 ng/ml and an intra- and interassay precision <8% and <10%, respectively. Lipopolysaccharide (LPS) was measured by ELISA (Cusabio, Houston, TX) with a sensitivity of 0.039 ng/mL and an intra- and interassay precision <8% and <10%, respectively.