1 n hcl

Deionized water, methanol (99.9%), and isopropanol (99.9%) were obtained from Fisher Scientific (Waltham, MA). 1 N NaOH, 1 N HCl, 5 N ammonium hydroxide, glacial acetic acid (99.99%), ultra-high-purity ammonium acetate (99.999%), total human serum IgG isolate (purity ≥95%, based on non-reduced SDS-PAGE and verified by nanoLC-MS/MS of tryptic digests), and BSF isolate (purity ≥90%) were purchased from Sigma-Aldrich (St. Louis, MO). Agencourt Cleanseq carboxyl-coated magnetic microparticles (COOH-beads) were from SCIEX (Brea, CA). PNGase F enzyme and bovine pancreas ribonuclease B isolate (purity ≥85%, based on SDS-PAGE) were from New England Biolabs (Ipswich, MA). A neodymium magnet was obtained from K&J Magnetics (Pipersville, PA). All bare-fused silica (BFS) capillaries (91 cm × 30 µm i.d. × 150 µm o.d.) with sheathless CESI-MS emitters in OptiMS cartridges were from SCIEX. A NanoBooster^TM unit for generating the DEN-gas was from Bruker Daltonics (Billerica, MA).

Total, MHV68-specific, and dsDNA immunoglobulin levels were determined as previously described (49 (link)). Briefly, Nunc Maxisorp plates (Fisher Scientific, Pittsburg, PA) were coated with antigen of interest, either anti-IgG (heavy and light) or anti-IgM antibodies (Jackson ImmunoResearch, West Grove, PA), UV-irradiated MHV68 virus stock in PBS (740,000 microjoules/cm² × 2) (Stratalinker UV Crosslinker 1800; Agilent Technologies, Santa Clara, CA), or dsDNA from Escherichia coli (12.5 μg/ml; Sigma-Aldrich, St. Louis, MO) overnight at 4°C. Plates were washed with PBS containing Tween (PBS-Tween) (0.05%) and blocked for 1 h with PBS-Tween (0.05%)-BSA (3%), incubated with fivefold serial dilutions of serum in PBS-Tween (0.05%)-BSA (1.5%) for 2 h and then washed with PBS-Tween (0.05%). Bound antibody was detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse total IgG (heavy and light chain [H+L]) or IgM (Jackson ImmunoResearch, West Grove, PA) using 3,3′,5,5′-tetramethylbenzidine substrate (Life Technologies, Gaithersburg, MD). HRP enzymatic activity was stopped by the addition of 1 N HCl (Sigma-Aldrich, St. Louis, MO), and the absorbance was read at 450 nm on a model 1420 Victor³V Multilabel plate reader (PerkinElmer, Waltham, MA).

Shrimp shell powder (160 g) was treated with 1 N HCl (Sigma-Aldrich, Saint-Louis, MO, USA) (1 L) for 2 h at 50 °C to remove minerals, and then filtered and washed until the pH of the filtrate was neutral. The filtered powder was dried at 200 °C for 4 h and digested further with 2.5 N NaOH for 2 h at 50 °C to remove proteins and other macromolecules, and then filtered and washed many times until a neutral pH. The chitin was dried in an oven at 80 °C for 24 h and bleached with 5% of NaClO (ACE, Pescara, Italy) 1:10 for 30 min before extracting chitosan.

All chemicals used for metabolite extraction and LC-TripleTOF experiments were ultra-pure LC-MS grade. Acetonitrile and methanol were procured from Honeywell Research Chemicals (Muskegon, MI, USA). Formic acid and 1N HCl were obtained from Sigma-Aldrich (St. Louis, MO, USA). Demineralized water (ddH2O) was generated by Millipore Advantage Milli-Q system (EMD Millipore, Billerica, MA, USA). Internal standards including: glucose-D7, glucose-6-phosphate-13C6, octanoic acid-D15, lauric acid-D23, tyrosine-D2, glutamic acid-D3, citric acid-D4, and chenodeoxycholic acid-D5 were purchased from Cambridge Isotopes Ltd. (Tewksbury, MA, USA). 25-hydroxycholesterol-D6 and lauroyl-l-carnitine-D3-(chloride) were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Valine D5, tryptophan D5, and cytosine-D2 were from CDN isotopes (Quebec, Canada). 1-oleoy-2-hydroxy-sn-glycero-3-phosphocholine-D7 was from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and caffeine-13C2 was from Toronto Research Chemicals (Toronto, Canada). Mass calibration solutions (APCI -Positive and Negative) were purchased from AB Sciex (P/N: 4460131 and 4460134; Concord, CA, USA).

A total of 600 μL Stem Pro 34 serum-free media alone or containing the relevant concentration of S1P (Cayman Chemicals) was placed in individual wells of a 12-well plate (Corning, London, UK) and 6.5-mm Transwell inserts (5-μm pore diameter) placed in each well. A total of 100 μL of cell suspension (1 × 10⁶ cells/mL) was added to the insert and incubated for 3 hours. Cell migration was assessed by quantification of the number of cells in each lower well using flow cytometry as described; migration was expressed as a percentage of the input cells in the lower well. S1P and FTY702-P were prepared by dissolving in 95% dimethyl sulfoxide/5% 1 N HCl (Sigma) and diluted for use in phosphate-buffered saline + 3% fatty acid free bovine serum albumin (Sigma). W146 (Sigma) was dissolved in methanol containing 0.05% acetic acid and diluted for use in cell culture media.

Acetone, 1N-HCl, SnCl₂, and sodium tartrate were purchased from Sigma Aldrich Korea (Seoul, Korea). Tc-99m pertechnetate was eluted from a commercial technetium generator (EnviroKorea, Daejeon, Korea) at our institution. A Radio-Cap®, capillary columns filled with silica materials (cat. FC-D1012, silica gel size = 38–75 μm; Futurechem, Seoul, Korea) were used for radio-chromatography. NCI-H460 (human non-small-cell lung cancer cell, wtEGFR-positive) and SW620 (human colon cancer cell, wtEGFR-negative) cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea).