Dulbecco s modified eagle s media dmem

MIA PaCa-2 and PANC1 cells were obtained from the ATCC (Manassas, Virginia, USA), whereas human pancreatic stellate cells, isolated from resected tumour tissue (HPSCs) were a kind gift from Professor David Yule (University of Rochester, New York, USA). BxPC-3 cells were a kind gift from Dr. Ayse Latif (University of Manchester, UK). All cell types were grown in 75-cm² culture-treated flasks (Corning, New York, USA) in high-glucose Dulbecco’s modified Eagle’s media (DMEM) or RPMI-1640 (Sigma Aldrich, Gillingham, UK). All media was supplemented with 10% foetal bovine serum (Biowest, Nuaillé, France) and 100-units/mL penicillin and 100μg/mL streptomycin (Sigma). All flasks were kept incubated at 37 °C and in 5% CO2. MIA PaCa-2 cells were discarded after being sub-cultured 30 times whereas HPSCs were discarded after 10.

Normal human cartilage specimens were extracted and separated from patients’ knee joints (n = 6) who had suffered above-knee amputations after severe lower extremity injuries. Human cartilage samples were taken from knee joints of OA patients (n = 6) who had underwent total knee arthroplasty (TKA). Southern Medical University’s Institute Research Ethics Committee reviewed and approved this study. Patients/participants gave their explicit written consent to take part in our research.
Primary chondrocytes were separated from the cartilage of the human tibial plateau and rinsed with Dulbecco’s modified Eagle’s media (DMEM; Sigma, St., USA) according protocol as previously described. Furthermore, cartilage tissue specimens were isolated and divided into fragments, before being treated with 0.25% trypsin for 30 min and 0.2% collagenase type II for 8 h at 37 °C (Sigma-Aldrich, USA). The digest was diluted in DMEM with 10% (v/v) fetal bovine serum (FBS) (Hyclone, USA), 1% penicillin/streptomycin (v/v) (Invitrogen, USA), 2 mM glutamine (Sigma-Aldrich, USA), as well as 50 g/mL ascorbic acid (Sigma-Aldrich, St. USA). Cells were plated in a Petri dish at a density of 1 × 10⁵ cells/mL as well as cultivated at 37 °C in an incubator of 95% O₂ and 5% CO₂. Cultivated chondrocytes were used in subsequent experiments when cells were at 80% confluence.

The mosquitoes were homogenized individually in 550 μL Dulbecco’s Modified Eagles Media (DMEM, Sigma-Aldrich, St Louis MO, US), 2% HEPES (Fisher Scientific, Fair Lawn, NJ, US). About 60 μL of ten individual mosquito homogenates (1×) were pooled to produce 10× pools (60 μL from 10 mosquito homogenates = 600 μL of 10× pool), and similarly, 60 μL from ten 10× mosquito homogenates were pooled together to make 100× pools (60 μL from ten 10× mosquito homogenates = 600 μL of 100× pool). The preparation of samples was performed at +4 °C, and subsequently stored at −80 °C.

Sheep infections were carried out essentially as described previously [32 (link)]. Briefly, >12 week-old Lleyn cross lambs were infected by oral administration of ~200 (parental clones and F1) or ~400 (F2) metacercariae per sheep. Infection status was monitored weekly by ELISA [117 (link)] from four weeks prior to infection and by faecal egg count (FEC) prior to infection and from eight weeks post infection. Treated sheep were dosed orally with TCBZ (Fasinex, Novartis), at the recommended dose rate of 10 mg/kg. At 12–16 weeks post infection, sheep were humanely euthanised and enumeration of adult liver flukes was performed by dissection of the bile ducts and incubation of the liver in PBS for 2 h at 37°C. Adult parasites manually recovered by dissection from the bile ducts, were washed in PBS, snap frozen and stored at -80°C. Eggs for downstream infection of snails (F1 and F2 eggs) were harvested from adult parasites purged by incubation in 1–2 ml of Dulbecco’s Modified Eagle’s Media (DMEM; Sigma-Aldrich, Dorset, UK) containing 1000 units penicillin, 0.1 mg streptomycin and 0.25μg amphotericin B (Sigma-Aldrich, UK) for a minimum of 2 h at 37°C.

HUVEC cell line was originally acquired from the American Type Culture Collection (Manassas VA, USA). Native LDL (nLDL) was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Dulbecco’s modified Eagle’s media (DMEM), dimethylsulfoxide (DMSO), and allicin were obtained from Sigma (St. Louis, MO, USA). LDH assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The AnnexinV-FITC kit was purchased from Invitrogen (Carlsbad, CA, USA). All other reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture reagents were purchased from Life Technologies [Trypsin-EDTA (0.05%), TrypLE, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), Anti-Anti, B-27 supplement along with Neurobasal, Dulbecco’s modified Eagle’s media (DMEM) and Minimum Essential Media (MEM) medias], Sigma-Aldrich [Cytosine β-D-arabinofuranoside (Ara-C), poly-D-lysine (70–150 kDa), Hank’s Balanced Salt Solution (HBSS), 1,4-Piperazinediethanesulfonic acid (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), insulin, N-Acetyl Cysteine (NAC), hydrocortisone, sodium pyruvate and GlutaMAX], Worthington (Trypsin, Papain, Papain Dissociation Kit, and DNase), Atlanta Biologicals [Horse Serum (HS) and Fetal Bovine Serum (FBS)]. Zinc indicators (FluoZin-3 AM and Newport Green DCF) were obtained from Life Technologies. Vitamin MEM solution, amino acid MEM solution, ZnCl₂ (0.1 M stock solution), N,N,N′,N′-Tetrakis (2-pyridylmethyl)ethylenediamine (TPEN), 2-Mercaptopyridine N-oxide sodium salt (pyrithione) were all purchased from Sigma-Aldrich. For GluA2 surface labeling, Alexa 488-CAM2 and Alexa 647-CAM2 were designed and synthesized by the Hamachi research group (Kyoto University; Wakayama et al., 2017 (link)).