Multiskan ascent microtiter plate reader

Frozen tissues stored in − 80 °C were weighed before cytokine extraction. Colon tissues were resuspended in T-PER Tissue Protein Extraction Reagent (ThermoFisher, Rockford, IL) and antifoam SE-15 (Millipore Sigma, St. Louis, MO). Then, tissues were homogenized using a Tissue-Tearor (Biospec Product, Inc, Model 985370-398). The homogenized solution was centrifuged for 10 min at 6000 rpm at 4 °C. The supernatant was recovered and stored at − 80 °C. A sample of the supernatant was also used to determine the protein concentration using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Rockford, IL). After cytokine extraction from the colon tissues, the samples were subjected to the mouse sICAM-1/CD54 Quantikine ELISA kit (R&D Systems, Minneapolis, MN) and the mouse TNF alpha uncoated ELISA kit (Invitrogen, Waltham, MA). ELISA protocols were followed according to manufacturer’s instructions. The sICAM ELISA assay was read with the Multiskan Ascent microtiter plate reader (Thermo Electron Corporation), and the TNF- $\alpha$ ELISA assay was read with the MQX200 UQuant microplate reader (BioTek).

Before lipid extraction, colon weights were measured so that hormone levels could be normalized to colon weight. Lipids were extracted from the colon to determine concentrations of estradiol and testosterone. To do this, a mixture of PBS (1x):HCl (0.1 N) (5:2 v/v) was added to each sample and then homogenized. Lipids were extracted with ethyl acetate:isopropanol (1:1 v/v) and then separated with PBS:ethyl acetate (3:2 v/v) mixture. The ethyl acetate phase was collected and evaporated using Speedvac at 30 °C for 2–3 h. Lipid samples were stored at − 80 °C until hormone measurements.
Testosterone and estradiol enzyme-linked immunosorbent assay (ELISA) kits were purchased from DRG and used to measure testosterone and estradiol levels in the colon, respectively. Lypocheck (Bio-Rad Laboratories) was used as a control for the DRG ELISA kits. Testosterone and estradiol assays were carried out according to the manufacturer’s protocol. ELISA plate absorbance values were read at 450 nm with the Multiskan Ascent microtiter plate reader (Thermo Electron Corporation).