Fitc conjugated lps

LPS (O111:B4) and FITC-conjugated LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). TLR4 Agonist-Ultrapure LPS (055:B5) and CpG-1826 were obtained from Invivogen (San Diego, CA, USA). The following fluorescence-conjugated antibodies (Abs) were provided by BioLegend (San Diego, CA, USA) and were used for flow cytometry analysis: anti-B220 (RA3-6B2), anti-CD11c (N418), anti-CD3 (17A2), anti-CD317 (927), anti-CD40 (3/23), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-IRF7 (MNGPKL), and anti-TLR4 (SA15-21). Anti-IFN-α (RMMA-1) Ab was purchased from pbl Assay Science (Piscataway, NJ, USA). Anti-class I major histocompatibility complex (MHC) Abs (28–8–6) and anti-class II MHC (M5/114.15.2) Abs were purchased from eBioscience (San Diego, CA, USA).

Fluorescein isothiocyanate (FITC)-conjugated LPS (1 μg/mL, Sigma-Aldrich, USA) was excited at 480 nm and monitored the changes in the emission of FITC-LPS at 515 nm in the incubation of different concentrations of peptides (0, 12.5, 25, 50, 100 μg/mL). Peptides were dissolved in 10 mM phosphate buffer at pH 6.0. The interactions between peptides and LPS were further assessed by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit (Xiamen Houshiji, China) following the kit instruction. Briefly, different concentrations of peptides (0, 12.5, 25, 50, 100 μg/mL) were incubated with LPS (1 μg/mL, Sigma-Aldrich, USA) at 37 °C for 30 min. After incubation, 100 μL TAL solution was added to 100 μL LPS-peptide mixtures in a pyrogen-free tube and incubated at 37 °C for 10 min. Then, LPS-peptide mixtures were added with pre-warmed substrate solution for additional 6-min incubation at 37 °C. Lastly, the absorbance at 545 nm was measured on a microplate reader (Epoch Etock, BioTek, USA). The percentages of LPS-neutralizing activity were calculated.

Goblet cells were sorted by using the BD FACSAria II sorter (BD Biosciences). Intestinal was separated, digested by 0.25% trypsin for 10 min, filtered through a nylon cell strainer (200 meshes), washed three times with PBS, and then incubated with MUC2 for 2 h to sort goblet cells.
Cells were cultured in 6-well plates and then 40 ng/ml FITC-conjugated LPS (No. F3665, Sigma) or linoleic acid was incubated for 24 h. The medium was discarded, the excess FICT-LPS was washed with PBS, and then cells transfected with FITC-LPS were collected and analyzed in a flow cytometer.
Cell apoptosis was measured by Annexin V-FITC/PI apoptosis detection kit (No. A211-01, Vazyme Biotech, china). Cells were seeded in six-well plate for one day and then were incubated with LPS, linoleic acid or SSO for 24 h. Cells (2.5 × 105 cells/well) were collected and cleaned with PBS, and then the cells were suspended in Annexin V binding buffer (100 μL) and stained with PI (5 μL) and Annexin V-FITC (5 μL). After the cells were incubated for 20 min in dark, 400 ul Annexin V binding buffer was added for the subsequent flow cytometric analysis.