Penicillin streptomycin

In vitro culturing of five different patient-derived spheroids was performed using fresh tumor tissue from five patients who underwent initial surgery for astrocytoma WHO grade III and IV from September 2007 through May 2008 at the Department of Neurosurgery, Odense University Hospital, Denmark. Written informed consent was obtained from all five patients. The tumor tissue was processed and cultured as described earlier [50 (link)]. In brief, tumor tissue was sectioned manually into small fragments of 200–400 μm using two scalpels. The tumor fragments were cultured for 10–15 days in 0.75% agar-coated culture flasks of 75 cm² containing 20 mL Dulbeccos Modified Eagle Medium (Sigma Aldrich, USA) supplemented with 10% fetal calf serum (Fisher Scientific, USA), 2% glutamine (Cambrex, USA), 4% nonessential amino acids (Cambrex), and 2% penicillin/streptomycin (Cambrex) until spheroids were formed in a standard tissue culture incubator (95% humidity, 95% air, and 5% CO₂). Commercial U87MG cell line-derived spheroids were cultured under the same conditions until reaching a size of 200–400 μm. Spheroids were then fixed in 4% neutral buffered formaldehyde for 24 h and embedded in paraffin. Afterwards, the preservation and expression patterns of TIMP-1, CD63, and the stem-related markers CD133 and Sox2 were investigated by immunofluorescence and confocal microscopy.

P3, 2012–018 and BG7 patient-derived GBM cells were expanded from biopsies obtained from patients undergoing tumour resection at the neurosurgery department (Haukeland University Hospital; Bergen, Norway). These cells, as well as long-term cell lines (U87, T98G, HF66 and A172), were propagated as monolayers in Dulbecco’s modified eagle medium (DMEM, Sigma-Aldrich; St. Louis, MO, USA) supplemented with 10% foetal bovine serum, non-essential amino acids, 100 U/mL penicillin/streptomycin and 400 μM L-glutamine (complete medium; all Cambrex; East Rutherford, NJ, USA) at 37 °C in a humidified atmosphere of 5% CO₂. Patient-derived cells and cell lines were authenticated by short tandem repeat loci (STR) analysis as described in Supplementary Methods. Profiles from patient-derived cells were compared with those from the original parental biopsy (P3, 2012–018 and BG7), and cell lines were authenticated against the profiles from previously fingerprinted stocks or manufacturer provided profiles, e.g., ATCC repository (T98G, A172 and U87) or manufacturer (NHA) Applied Biological Materials Inc. (Richmond, BC, Canada).

The phagocytosis assay was performed as previously described [37 (link)]. In brief, the retinas of non-dystrophic RCS rats at age 8 weeks were detached under dim red light. Photoreceptors of the isolated rat retinas were facing the RPE monolayers in Neurobasal A medium supplemented with 1% N2, 2% B27, 2 mM Glutamax (all from Gibco, Life Technologies), and 100 U/mL Penicillin/Streptomycin (Cambrex). After co-cultured with rat retinal explants for two days at 37°C, 5% CO₂, the retinal explants were removed and 3D-RPE and SD-RPE cells were analyzed by immunostaining. The internalization of rat POS by RPE cells was immunostained using anti-Rhodopsin antibody (ab81702, 1:200, abcam), while filamentous actin was detected with FITC-conjugated phalloidin (P5282; Sigma-Aldrich) to demonstrate the cell morphology. In Z-axis scan model, we used Zen lite 2012 software and Zeiss LSM 700 confocal microscope to show the location of internalized POS. For quantitative calculation, 4 random fields of view (135 μm × 180 μm) were photographed per group and the number of internalized POS was counted. Three independent experiments were conducted.

FLS were successfully isolated from RA (n = 6), OA (n = 6) and healthy (n = 6) synovial biopsies subjected to a mild proteolytic treatment with 0.05% trypsin and 0.5 mM EDTA in phosphate-buffered saline (PBS) for 10 minutes at 37 °C under gently shaking. Trypsin was then neutralized with fetal calf serum (FCS) (Celbio, Milan, Italy) and cells were plated in culture dishes (3 cm diameter, BD Falcon, BD Biosciences, San Jose, CA, USA) at a concentration of 1–1.5 × 10⁶ per dish in RPMI 1640 medium (Cambrex Bio Science, Milan, Italy) supplemented with 15% FCS, 2 mM glutamine and penicillin–streptomycin (Cambrex Bio Science). Once at confluence (about 3 weeks), the cell monolayers were expanded in culture by splitting 1:2 every 7 days. The cells were considered FLS (type B synoviocytes) if negative by immunostaining with anti-CD68, anti-CD14, anti-CD11b and anti-CD11c antibodies (markers of type A macrophage-like synoviocytes), positive by staining for the enzyme uridine diphosphoglucose dehydrogenase (preferentially expressed by the intimal lining FLS and reflecting their ability to synthesize hyaluronan, an important constituent of synovial fluid)^{3 (link)}, and if they had a spindle-shaped, fibroblast-like morphology ( > 95% cell purity). Cell monolayers were used within the seventh passage in culture.

Vero cells were cultured in 6-well plates until 100% confluent. The virus was treated with 75 µM EGCG or EGCG-S for one hour. The untreated virus served as the control. Virus dilutions ranging from 10⁻³ to 10⁻⁷ were prepared. Cells were infected with the diluted virus for one hour and the unadsorbed virus was aspirated. Plaque assay method is a modification of protocol previously reported in Adams et al. [42 (link)]. After one hour of incubation, cells were overlaid with plaque media consisting of bacteriological agar containing 3X Eagle medium (Gibco Invitrogen Corporation, Grand Island, NY, USA), 1.5 mL of 5% sodium bicarbonate (Gibco Invitrogen Corporation, Carlsbad, CA, USA), 0.5 mL FBS, 0.1 mL DEAE-dextran (ICN Biomedicals Incorporated, Aurora, OH, USA), 0.1 mL penicillin/streptomycin (Cambrex, Walkersville, MD, USA), with 0.05 mL gentamicin and 0.6% bacteriological agar (Oxoid Limited, Baskingstoke, Hampshire, England). Both solutions were placed in a 41 °C water bath, combined 1:1, and cells were overlaid with 3 mL of plaque media.
After 72 h of incubation at 5% CO₂, cells were stained with crystal violet and plaques were counted followed by the calculation of mean and standard deviation.

Cell culture reagents used were Penicillin-Streptomycin, RPMI 1640 obtained from Cambrex (Belgium) and fetal bovine serum from Life Technologies Ltd (UK). The TLR ligands used were chloroform extracted Escherichia coli (E.coli) lipopolysaccharide (LPS) and resiquimod (R-848) from Invivogen (USA). Flagellin (purified) and Pam₃cys-ser(lys)₄.3HCl (Pam3) were from Alexis (UK). Mianserin hydrochloride was purchased from Sequoia Research Products (Pangbourne, UK). The structural derivatives of mianserin were synthesized by Oxygen Healthcare Research Pvt. Ltd. (Ahmedabad, India), the synthesis steps are provided in the Supplemental Information. Macrophage colony stimulating factor (M-CSF) was purchased from eBioscience (USA). Dimethyl sulfoxide (DMSO) and Hank's balanced salt solution (HBSS) without calium chloride and magnesium sulfate were purchased from (Sigma, UK). Percoll Plus was purchased from GE Healthcare (Bucks, UK), PBS citrate from Paris Anticorps (France) and lympholyte-H from CedarLane (Ontario, Canada).

The PC-3 cells, an androgen resistant, p53-negative and Kirsten-Ras (K-Ras) mutated human prostate cancer cell line and the DU-145 human prostate cancer cell lines were obtained from American Type Cell Culture (ATCC, Manassas, VA, USA). Low passage (passage 10–15) PC-3 cells were maintained in Dulbecco’s modified Eagle’s medium (Cambrex, Walkersville, MD, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Biochrom, Berlin, Germany) and 100 U/ml penicillin/streptomycin (Cambrex, Walkersville, MD, USA). Low passage DU-145 cells were maintained in Minimum essential media supplemented with FBS and 100 U/ml penicillin/streptomycin (all from GIBCO BRL, Invitrogen Corp, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2.
Subcutaneous injections in SCID mice.
Male SCID mice (7 weeks old) were obtained from Democretos Laboratory. Mouse handling and experimental procedures were approved by the Hellenic Ministry of Rural Development and Food, General Directorate of Veterinary. Animal handling and experimental procedures were obtained in the Experimental Surgery Laboratory of the Athens Medical School. Implantations were carried out as previously described (Armakolas et al., 2015 (link)). Briefly, a suspension of 1 X 107 cells in 200 μl in 1X PBS was injected subcutaneously in each mouse.