Dulbecco s phosphate buffered saline pbs

Methanol for sample preparation and HPLC-grade solvents were purchased from VWR International (Edmonton, AB, Canada). Pure curcumin standard (Product No. 78246, ≥99.5%), was purchased from Sigma-Aldrich (St. Louis, MO, USA). Deuterium-labelled curcumin standard, namely d₆-curcumin, was purchased from C/D/N Isotopes (Pointe-Claire, QC, Canada). Most chemicals and reagents that were required for chemical- and cell-based antioxidant assays and cell viability assays were purchased from Sigma-Aldrich, while the glutathione Assay Kit was purchased from Cayman Chemical (Item No. 703002; Ann Arbor, MI, USA). Caco-2 cells used in this study, were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Cell culture dishes and plates were purchased from Sarstedt AG & Co. KG (Nümbrecht, Germany). Dulbecco’s modified Eagle’s medium (DMEM; Product No. D5796), Dulbecco’s Phosphate Buffered Saline (PBS; Product No. D8537), Penicillin-Streptomycin (Product No. P0781) came from Sigma-Aldrich. Fetal Bovine Serum (FBS; Product No. 10437028) and Trypsin-EDTA (0.05%), phenol red (Product No. 25300062) produced by Gibco™ were purchased from Fisher Scientific.

HN30 cells were utilized in this assay because of high levels of AHR expression. Cells were seeded into 12-well plates at 50,000 cells/well and cultured for 48 h in DMEM/F12 medium (Sigma) with 10% fetal bovine serum (Gemini BioProducts, West Sacramento, CA, USA), 25 mM HEPES, pH 7.4, and 100 U/mL penicillin and 100 μg/mL streptomycin. The medium was removed, and cells were washed with Dulbecco’s phosphate-buffered saline (PBS, Sigma). Cells in each well were incubated in 500 μL Hanks balanced salt solution (Gibco), 25 mM HEPES (pH 7.4), and 5 mg/mL bovine serum albumin for 30 min in an incubator. Next, cells were treated with AHR ligands, followed by the addition of 2 pmoles of [¹²⁵I]-PAL, and cells were placed back in the incubator for 30 min. The medium was removed, 500 μL of PBS was added, and cells were exposed to UV light at >302 nm, at a distance of 8 cm, for 4 min using two 15-W UV lamps (Dazor Mfg. Corp. St. Louis, MO, USA). The PBS was removed, and cells lysed in 100 μL of 25 mM MOPS, 2 mM EDTA, 0.02% sodium azide, 10% glycerol, and 1% NP40. Lysates were transferred to microfuge tubes and centrifuged at 18,000× g for 20 min. Supernatants were subjected to tricine SDS-PAGE, and subsequently transferred to PVDF membrane. The radioactive AHR band was visualized with X-ray film, excised, and counted in a gamma counter.

4-vinylbenzyl chloride (4VBC), glycidyl methacrylate (GMA), methacrylic anhydride (MA), triethylamine (TEA), 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), 2,4,6-trinitrobenzenesulfonic acid (TNBS), acetic acid (AcOH), and Dulbecco’s Phosphate Buffered Saline (PBS) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used as received. Rat tails were received from Central Biomedical Services at the University of Leeds (UK) and used for the acidic extraction of type I collagen. Dulbecco’s modified Eagle’s medium (DMEM), foetal calf serum (FBS) and penicillin-streptomycin (PS) were purchased from Gibco (Waltham, MA, USA). All the other chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA).

Platelets in whole blood were analyzed for ACKR3 platelet surface exposure gating for the platelet-specific marker CD42b. Blood collected in citrate phosphate dextrose adenine was diluted 1:50 with Dulbecco’s phosphate-buffered saline (PBS; Sigma Aldrich Co., St. Louis, MO, USA) and incubated with the respective conjugated antibodies, mouse monoclonal anti-human ACKR3-PE (R&D systems, Minneapolis, Minnesota, USA) and mouse anti-human CD42b-FITC (Beckman Coulter, Brea, California, USA) or their respective isotype controls (R&D systems) for 30 min at room temperature (RT). After staining, the cells were fixed with 0.5% formaldehyde and analyzed by flow cytometry (FACS-Calibur flow cytometer Becton-Dickinson, Heidelberg, Germany).

IFP tissue (5–10 cc) was mechanically dissected and washed repeatedly with Dulbecco’s Phosphate Buffered Saline (PBS; Sigma), followed by enzymatic digestion using 235 U/mL Collagenase I (Worthington Industries, Columbus, OH, USA) diluted in PBS and 1% bovine serum albumin (Sigma) for 2 h at 37 ℃ with agitation. Cell digests were inactivated with complete media [DMEM low glucose GlutaMAX (ThermoFisher Scientific, Waltham, MA, USA) + 10% fetal bovine serum (FBS; VWR, Radnor, PA, USA)], washed, and seeded at a density of 1 × 10⁶ cells/175 cm² flask in complete human platelet lysate (hPL) medium. Complete hPL medium was prepared by supplementing DMEM low glucose GlutaMAX with hPL solution and 0.024 mg/mL xeno-free heparin (PL Bioscience, Aachen, Germany) to obtain a 10% hPL final concentration. IFP-MSC were cultured at 37 °C 5% (v/v) CO₂ until 80% confluent as passage 0 (P0), then passaged at a 1:3 ratio until P2, detaching them with TrypLE™ Select Enzyme 1× (Gibco, ThermoFisher Scientific, Waltham, MA, USA) and assessing cell viability with 0.4% (w/v) Trypan Blue (Invitrogen, ThermoFisher Scientific).