Spectramax m5e fluorescent microplate reader

Insulin, leptin, LPS and IL-6 levels were estimated in serum collected after sacrifice.
Respective ELISA kits (insulin, leptin, IL-6 -MilliPlex Map Mouse Adipokine kit, Merck, Germany; LPS -E2214Mo, Bioassay Technology Laboratory, UK) were used as per the manufacturer's instructions. Colorimetric readings were taken using SpectraMax m5e fluorescent microplate reader (Molecular Devices, USA) or MAGPIX multiplex system (Merck, Germany).

Intestinal permeability tests were performed during the 11 th and 12 th weeks of the study. 600 mg/kg body weight Fluorescein isothiocyanate-dextran (average mol wt. 3,000-5,000, Sigma-Aldrich, Missouri, USA) in 0.9% saline was given to mice orally. Animals were subjected to hair removal and fluorescence imaging (IVIS Lumina Series III, Perkin Elmer, USA) of the abdominal region at 0 (before treatment), 30, 60, 90 and 120 min after treatment, under anesthesia (Isoflurane: induction at 4%, maintenance at 2.5-3%). Blood was collected before and 90min after treatment, and serum was separated by centrifugation at 8000rpm for 10min.
Fluorescence in serum was measured using excitation at 485nm and emission at 530nm (SpectraMax m5e fluorescent microplate reader, Molecular Devices, USA). After sacrifice, colon samples were stored in either 10% formalin (in PBS) or Carnoy's fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) for further processing. For paraffin embedding, tissues were prepared by serial dehydration in ethanol (25%, 50%, 70%, 90%, 100%, 2 h each) followed by xylene treatment (2h, twice). After embedding (in molten paraffin at 60°C, 2 h, twice) and microtomy blocks formation, 5µm thick sections were made.

HEK-293 cell line was procured from National Centre for Cell Science, Pune, India. Cells were maintained in DMEM (Lonza, Switzerland) containing 10% FBS (Lonza, Switzerland) and 1X
PenStrep (Thermo Fisher Scientific, USA) at 37°C, 5% CO2. All experiments were performed between passage numbers 25-35. Plasmid pVQ CMV NanoV1-2a-EGFP ferritin was purchased from Addgene, USA (Addgene plasmid #79649, deposited by Jeffrey Friedman) [26] (link). Cells were seeded in 96 well plates, and at 70-80% confluency, transfection was carried out using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, USA), as per supplier's instructions (0.2µg DNA/well, 0.4µl transfection reagent/well). After 36-48h, transfection was verified by GFP visualization and TRPV1 activity (through Ca 2+ -influx measurement) was confirmed using capsaicin (50µM). Fura-2AM (Thermo Fisher Scientific, USA) fluorescent dye was used for intracellular Ca 2+ measurements. HEK cells were incubated in a medium containing 10µM Fura-2AM for 45min at 37°C. Then, media was removed and respective treatments were given (n=6-8 each). Immediately, the cells were subjected to fluorimetry (SpectraMax m5e fluorescent microplate reader, Molecular Devices, USA) at excitation 340 nm and 380 nm, emission at 510 nm, through 2 min at 20s intervals). F340/F380 ratio was interpreted as intracellular Ca 2+ levels.