Dy453

Pre-adipocytes were plated at a density of 200,000 cells/12-well. The next day, cells were exposed to CRF peptides plus/minus LPS at different time points and cell culture supernatants were collected and stored at −80 C until used for the determination of adipokine and interleukin concentration by ELISA.
Parallel experiments were performed in fully differentiated adipocytes. In brief, pre-adipocytes were cultured to 12-well plates at a density of 20,000 cells per well and forced to differentiate as mentioned above. Then, fully differentiated adipocytes were exposed to CRF peptides plus/minus LPS at different time points and cell culture supernatants were collected and stored at −80 C. Cell culture supernatants were also collected from adipocytes exposed to CRF peptides plus/minus LPS during the differentiation process.
Adiponectin was measured by MRP300 and Duoset (DY1119), while Leptin was measured by MOB00 and Duoset (DY498) purchased from R&D (R&D, NE). ELISA assays for IL-6 (DY406), CXCL1/KC (DY453), TNF-α (DY410), IL-1b (DY401) and IL-10 (DY417) were all purchased from R&D. For normalization of the measurements, cells were harvested and sonicated for quantification of total cellular proteins as previously described [14] (link).

All uterine lavage samples were frozen at −80°C until analysis. For experiment 1, only CXCL1 (mouse KC) was assessed using a commercial ELISA kit (DY453; R&D Systems; Minneapolis, MN) exactly as per the manufacturer's instructions. For experiment 2, all samples were analyzed for cytokine and chemokine content using the Luminex multiplex system (Milliplex MAP kit, mouse cytokine/chemokine catalog #MPXMCYTO-70K; Merck-Millipore, Kilsyth, VIC, Australia) with a panel of 15 targets (CSF1, CSF2, CSF3, IL1A, IL1B, IL6, IL10, CXCL1, CXCL10, LIF, CCL2, CCL3, CCL4, CCL5, and TNFα). The manufacturer's protocol was followed precisely.

Adapted from Chamberlain et al., 2019 [3 (link)]. Lung protein samples were assayed for IL-6 (DY406, R&D, Minneapolis, MN, USA), GCSF (DY414, R&D, Minneapolis, MN, USA), IL-1β (DY401, R&D, Minneapolis, MN, USA), KC (DY453, R&D, Minneapolis, MN USA), IFN-β (DY814-05 and DY8234-05, R&D, Minneapolis, MN, USA), and CCL20 (DY760, R&D, Minneapolis, MN, USA) by ELISA according to the manufacturer’s instructions.

The liberation of tumor necrosis factor α (TNFα), interleukin-6 (IL-6), CXCL1, and CXCL2/3 was measured in the BAL of mice three hours after LPS exposure by ELISA (DY410; DY406; DY453; DY452; R&D Systems; Minneapolis, MN, USA) according to the manufacture’s protocol.

For inhibitor screen, human BJ fibroblast cells (ATCC CRL2522) (American Type Culture Collection, VA) were used. For mouse cell based assay MLE-12 mouse epithelial cells (ATCC CRL2110) were used. Both cell lines were maintained in ATCC recommended media. Cells were seeded at 5 × 10³ cells/well into 96-well flat-bottom microtiter plates in which peptides that had been pre-diluted with cytokines (1 ng/mL for human IL-17A or 15 ng/mL for mouse IL-17A) in culture medium. Cells were incubated at 37 °C for 16–24 hrs, and supernatants were collected and analyzed by ELISA for either human CXCL1/GRO-α (R&D Systems DY275) or mouse CXCL1/KC (R&D Systems DY453).

Differentiated podocytes were cultured in 75 cm² flasks and were re‐seeded in 96‐well plates for 24 h, at 6000 cells/well. All experiments were performed under serum starvation conditions (RPMI‐1640, with 0.2% heat‐inactivated FBS and 5 mmol/L glucose) including a 16‐h preincubation to synchronize the cells. MCP1 and KC expression was quantified using Duoset ELISA kit according to manufacturer's protocol (DY479 for MCP‐1; DY453 for KC, R&D Systems purchased from Zug, Switzerland).