Sodium bicarbonate solution

The murine macrophage cell line, RAW264.7 was cultured at 37 °C/5% CO₂ in DMEM containing 10% inactivated fetal bovine serum (FBS, WELGENE, Gyeongsan, Republic of Korea, S 001-01), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, 15140-122). The human colon epithelial cell line, Caco-2 was cultured at 37 °C/5% CO₂ in DMEM/F-12 (Gibco, 11320033) containing 20% inactivated FBS, 1% MEM nonessential amino acid solution (Gibco, 11140-050), 10 mM HEPES (Gibco, 15630-080), 0.1% sodium bicarbonate solution (Gibco, 25080-094), 10 μg/mL of gentamicin, 100 U/mL penicillin, and 100 mg/mL streptomycin. When the cells were about 80% confluent, they were detached from the culture dish and used for experiments.

Coupon recovery liquid was assayed by thawing samples at room temperature and then serially diluted (1 in 10) with MEM (Gibco), 1% l-glutamine (Gibco), 1% nonessential amino acids, and 2.5% 1 M HEPES. A volume of 100 μl of each dilution was pipetted in duplicate (technical replicates) for up to four replicates for neat dilutions (400 μl) onto confluent Vero E6 cells within a 24-well plate (7.9 × 10⁴ cells/cm²). After 1 h of incubation (±15 min) at 37°C with plate rocking every 15 to 20 min, 0.5 ml CMC overlay was added to each well, containing 1.5% CMC (3% [wt/vol] carboxymethylcellulose solution in sterile distilled water [Sigma C4888]), 1% antibiotic antimycotic solution (100×) (Sigma-Aldrich), 2× overlay medium (20% 10× MEM [Gibco]), 2% l-glutamine (200 mM) (Gibco), 2% nonessential amino acids (Gibco), 6% sodium bicarbonate solution (Gibco), 8% fetal bovine serum (Sigma-Aldrich), 5% HEPES buffer (Gibco), and 57% distilled water (Versol). After 3 days of incubation at 37°C, cells were fixed with formaldehyde and stained by addition of approximately 250 μl 0.2% crystal violet for 5 min before washing with water. The number of plaques in each well was determined and expressed as plaque-forming units.

Human RPE cells were grown in DMEM/F12 (Corning DMEM Hams F-12 50/50 Mix), supplemented with 10% of fetal bovine serum and 3% of sodium bicarbonate solution (Gibco) at 37°C with 5% CO₂. Prior to infection, 2.5 × 10⁵ cells⋅ ml^–1 of RPE were seeded in 24-well plates to reach ∼80% confluence (microscopically verified) in 24 h. Media were gently aspirated from the top of the cells, and each well was washed using antibiotic-free media. Overnight LB liquid culture-grown bacteria were diluted in DMEM/F12 to achieve the multiplicity of infection (MOI) of 50:1. Infected cells were then incubated at 37°C for 24 h. Bacterial numbers were enumerated by plating serial dilutions onto LB agar and counting colonies at 0 (to verify MOI) and 24 h post-infection.

Capsaicin and C75 were obtained from Sigma Chemical Co. (St. Louise, MO, USA). Eagle's Minimum Essential Medium (EMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, sodium pyruvate, L-glutamine, sodium bicarbonate solution, non-essential amino acid solution, and PBS tablets were obtained from Gibco BRL (Grand Island, NY, USA). Propidium iodide molecular probe were purchased from Life Technology (Invitrogen, Grand Island, NY, USA). M-PER mammalian protein extraction reagent, BCA protein assay reagent (bicinchoninic acid), polyvinylidenedifluoride membranes (PVDF) membrane, and Halt Protease Inhibitor Cocktail were purchased from Thermo Scientific (Rockford, IL, USA). Anti-fatty acid synthase (FASN) and anti-β-actin antibodies were purchased from Abcam (Biomed Diagnostics (TH) Co., Ltd, Thailand). Anti-ACC and anti-ACLY were purchased from Merck Millipore (Darmstadt, Germany) and Cell Signaling Technology Inc (Boston, MA, USA), respectively. All other chemicals and reagents were the highest grade available.

Small intestine segments were prepared as above (mIHC) and subjected to a FFPE technique. Slides were prepared in 4-μm sections and dewaxed with xylene (Cat# X5-4; Thermo Fisher Scientific; 3 × 10 min), rehydrated through a graded series of ethanol solutions (100% 3 × 10 min; 95% 2 × 5 min; and 70% 1 × 10 min), and washed in distilled water (1 × 5 min). Samples were stained with hematoxylin (1 × 10 min; Cat# SH26-500D; Thermo Fisher Scientific) and washed in distilled water again (1 × 10 min). Samples were then dipped into acid alcohol (two to five times) before being washed in distilled water (1 × 10 min). They were then dipped into sodium bicarbonate solution (Cat# BP328-1; Thermo Fisher Scientific) 20 times. Samples were then washed in water (1 × 10 min), and dehydrated through a graded series of ethanol solutions with eosin staining in the order: 70% alcohol (20 dips), eosin (1 × 3 min), 95% alcohol (2 × 20 dips), 100% alcohol (3 × 5 min). Lastly, the slides went through xylene (4 × 5 min) and slips were covered. All slides were scanned on the Aperio AT scanning system (Leica) using appropriate exposure times.