Nasal polyp fibroblasts were obtained from surgical samples in patients operated for CRSwNP. Polyps were cut into 2 mm3 pieces, placed in a trypsin solution for 30 min (Tryspin-EDTA 0.05% phenol red, Thermo Fisher Scientific, Waltham, MA, USA), passed through a 100 μM corning cell strainer and resuspended in culture medium (Dulbecco’s-modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 10% fetal calf serum (FCS), 10 μg/mL gentamycine, 10 µg/mL ceftazidime and 2.5 µg/mL amphotericin B (all from Thermo Fisher Scientific, Waltham, MA, USA)). For the quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis, cells were used until the 3rd passage. For immunofluorescence studies, fibroblasts were cultured on 10 mm diameter glass coverslips. For the RT-qPCR, Western blot analysis and immunofluorescence, cells were starved for 24 h in DMEM supplemented with 0.5% FCS before being treated with or without 10 ng/mL recombinant human TGF-β1, IL-4 (PeproTech, Rocky Hill, NJ, USA) or OSM (R&D Systems Europe, Lille, France), alone or in combination for 6 h for immunofluorescence, 24 h for mRNA quantification or 48 h for Western blot analysis.
Gentamycine
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Gentamycine is a broad-spectrum antibiotic used in microbiology laboratories. It is effective against a variety of gram-negative and some gram-positive bacteria. Gentamycine is commonly used for bacterial identification and susceptibility testing.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Round bottom 96 well plate, by Greiner (2 mentions)
Amniomax c 100 complete medium, by Thermo Fisher Scientific (2 mentions)
Ceftazidime, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Tryspin edta, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Coulter counter, by Beckman Coulter (1 mentions)
Dermatome, by Zimmer Biomet (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Mcf 7 human breast cancer cell line, by American Type Culture Collection (1 mentions)
12 protocols using gentamycine
1
Isolation and Culture of Nasal Polyp Fibroblasts
2
Isolation and Stimulation of PBMCs
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Mononuclear cells from peripheral EDTA whole blood (PBMCs) and heparinized bone marrow (BM-MNCs) were isolated with Ficoll-Paque (GE healthcare, UK) density gradient separation. Cells were washed twice with phosphate buffered saline (PBS), counted in a Coulter counter (Beckman Coulter, USA), and brought to concentration in Dutch modified RPMI medium (Roswell Park Memorial Institute; Invitrogen, CA, USA), supplemented with 50μg/ml gentamycine, 2 mM Glutamax (Thermo Fisher Scientific, USA) and 1 mM pyruvate (Thermo Fisher Scientific, USA). 5 x 105 PBMCs were cultured in a final volume of 200 μl / well in round bottom 96-well plates (Greiner Bio-one, Austria) and stimulated with RPMI, heat-killed Candida albicans (1 × 106 / ml, strain UC820). After 24 hours supernatants were collected and stored at -80°C until measurements were performed.
3
Tumor Tissue Dissociation and Cell Culture
4
Tumor Tissue Dissociation and Cell Culture
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Tumor pieces were collected into DMEM (Dulbecco’s Modified Eagles Medium, PAA Laboratories GmbH, Pasching, Austria). Biopsies were cut into 1 mm3 pieces and placed into either DMEM for immediate processing or into freezing medium (90% serum, 10% DMSO) prior to being progressively cooled to −80°C. Cell dissociation was performed mechanically by passage through increasingly finer needles (19G to 26G). Single cells were seeded in AmnioMAX™ C-100 complete medium containing gentamycine, L-glutamine and FBS (Gibco, Invitrogen, Paisley, UK) and maintained at 37°C in a 5% CO2 humidified atmosphere. The cells were further cultured until appearance of adherent cells and colony formation and then weekly passaged.
5
Isolation of Migratory Langerhans Cells
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Human skin tissue was obtained from healthy donors undergoing corrective breast or abdominal surgery after informed consent in accordance with our institutional guidelines. Split-skin grafts of 0.3 mm were harvested using a dermatome (Zimmer). The slides were incubated with Dispase II (1 U/ml) for 1 hour at 37°C and subsequently the epidermis was mechanically separated from the dermis. Migratory LCs were generated by floating the epidermis onto Iscoves Modified Dulbeccos’s Medium (IMDM) supplemented with 10% FCS, gentamycine (20 μg/ml, Centrafarm), pen/strep (10 U/ml and 10 μg/ml, respectively; Invitrogen) for 2 days. The emigrated cells were layered on a Lymphoprep (1.077 g/ml, Axis-shield) gradient and were routinely 95% pure and expressed high levels of langerin and CD1a. THP-langerin and the CD34+ human AML cell line MUTZ3 (MUTZ-LCs) were generated and cultured as described before [3 (link)].
6
Isolation and Expansion of Human Myoblasts
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Human myoblasts were isolated from the quadriceps muscle of a 5-day-old infant in accordance with the French legislation on ethical rules, as previously described [13 (link)]. Cells were expanded in a growth medium consisting of 199 medium and DMEM (Invitrogen, Life Technologies, Saint-Aubin, France) in a 1:4 ratio, supplemented with 20% foetal calf serum and 50 μg/ml gentamycine (Invitrogen), at 37°C in a humid atmosphere containing 5% CO2. Population doublings (PDs) were determined at each passage according to the formula: log (N/n)/log 2 where N is the number of cells counted and n is the number of cells initially plated.
7
Differentiation of Pro/Pre-B Cells into sIgM+ Cells
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For differentiation into surface IgM-expressing (sIgM+) cells, 3 × 104 FACS-purified BM-derived B220+c-kit+CD19+ Pro/Pre-B I cells were seeded in 96-well round-bottom plates (Greiner bio-one GmbH, Frickenhausen, Germany) and cultured at 37°C and 5% CO2 in 200 μl Iscove's Modified Dulbecco's Media (IMDM) supplemented with 10% (v/v) FCS, 1 mM sodium pyruvate, 1 mM HEPES, 2 mM Glutamax, 100 U/ml Penicillin-Streptomycin, 0.1 mg/ml Gentamycine, 0.1 mM nonessential amino acids, and 0.55 mM β-mercaptoethanol (all Invitrogen) for up to 4 days, in the absence of added IL-7. Surface IgM expression was analyzed using flow cytometry.
8
Culturing MCF-7 Breast Cancer Cells
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MCF-7 human breast cancer cell line was obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Cells were maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 μg/mL gentamycine (Invitrogen, Life technology), and 2 mM L-glutamine (Invitrogen, Life technology). Cells were cultured in 75 cm2 culture flasks at 37°C in a humidified atmosphere of 5% CO2.
9
Immortalized B-cells from A-T Patients
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Peripheral blood mononuclear cells (PBMCs) were obtained from A-T patients and healthy donors by Ficoll-Hypaque (Biochrom, Berlin, Germany) density gradient centrifugation. Lymphoblastoid B-cell lines (BCLs) were generated by Epstein Barr Virus (EBV) immortalization of patients' and healthy donors' PBMCs using standard procedures, and were grown in RPMI-1640 (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, California), 2 mmol L-glutamine (Gibco, Carlsbad, California), 50 g/ml gentamycine (Gibco, Carlsbad, California), 10% penicillin-streptomycin (Lonza, Verviers, Belgium), and cultured at 37 °C, 5% CO 2 . Serum starvation was induced incubating the cells in medium without FBS for 2 h. All the experiments were approved by the Ethical Committee "Comitato Etico per le Attività Biomediche Carlo Romano" of the Federico II University of Naples.
10
Coculture of Sorted Immune Cells
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Sorted Tfh cells, non-Tfh CD4 + T cells and purified CD19 + B cells were cocultured at 50 000 cells/well in 96-well plates (Starstedt) at a 1:1 ratio in 200 µL of TexMACS medium (Miltenyi Biotec) supplemented with gentamycine (Gibco) and 0.1 µg/mL SEB (Toxin Technology, Sarasota, Florida, USA) from 13 patients with SSc (3 dSSc and 10 lSSc) and 6 HC. Supernatants were collected after 6 days of culture. For blocking experiments, 10 µg/mL recombinant Human IL-21 Fc Chimaera Protein (IL-21RFc) (R&D Systems, Lille, France), or 10 µg/mL anti-IL6R-IgG1 (Roactemra, tociluzimab; Roche), or 0.1 µg/mL ruxolitinib (InvivoGen, San Diego, California, USA) were added in the culture. The total IgG and IgM levels were measured in coculture supernatants by ELISA (IgG Total Human uncoated ELISA kit and IgM human uncoated ELISA kit; ThermoFisher) according to the manufacturers' instructions.
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