Panc-1 adenocarcinoma cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and was cultured in Dulbecco’s modified Eagle medium (DMEM with 4.5 g/L glucose, BE12-604Q; Lonza, Basel, Switzerland), and supplemented with 10% fetal bovine serum (FB-1090; Biosera, Nuaille, France) and 0.4% gentamycin (Sandoz, Basel, Switzerland; 80 mg/2 mL). Cells were kept at 37 °C in a humidified atmosphere of 5% CO2. Cells were tested for Mycoplasma sp. applying the methodological article written by Uphoff et al. [54 (link)]. Only negative cell line was used for research purposes.
Gentamycin
Manufactured by Sandoz
Sourced in France, Austria
Gentamycin is a laboratory equipment product used for the detection and quantification of the antibiotic gentamicin in various samples. It serves as a tool for researchers and scientists working in the field of antimicrobial analysis and pharmaceutical quality control.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Merck Group (3 mentions)
Be12 604q, by Lonza (2 mentions)
Fb 1090, by Biosera (2 mentions)
L glutamine, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Ibitreat microscopy chamber, by Ibidi (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Rpmi 1640 medium, by Biosera (1 mentions)
Optima max xp benchtop ultracentrifuge, by Beckman Coulter (1 mentions)
Mla 55 fixed angle rotor, by Beckman Coulter (1 mentions)
Milli q gradient a10 system, by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Hydroethidine, by Merck Group (1 mentions)
Scintillation fluid, by PerkinElmer (1 mentions)
Ethanol, by Lach-Ner (1 mentions)
3h thymidine, by Moravek Biochemicals (1 mentions)
Mccoy s 5a modified medium, by Merck Group (1 mentions)
Ham s f 12 nutrient mix, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Rpmi1640 lm r1640, by Biosera (1 mentions)
8 protocols using gentamycin
1
Culturing Panc-1 Adenocarcinoma Cells
2
HT29 Cell Culture for Microscopy
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HT29 human colorectal cancer cells (ATTC_HTB-38) were grown in triplicates for 24 h on 8-well Nunc Lab-Tek (177,402, Nagle Nunc, Rochester, USA) for confocal microscopy and 8-well Ibitreat microscopy chamber (Ibidi GmbH, Martinsried, German) for STED microscopy (initial cell number: 5000 cell/300 µl/well). For electron microscopy (EM) and DAB immune EM, we cultured the cells on Lab-Tek chamber (initial cell number: 8000 cell/300 µl/well) for 72 h in RPMI 1640 medium (Biosera, Ringmer, UK) supplemented with 2 mM L-glutamine (Merck-Sigma-Aldrich, Darmstadt, Germany), 80 mg/2 ml gentamycin (Sandoz) and 10% sEV-depleted FBS. To preserve the MVB-like sEVCs, we decanted the culture medium carefully from the cultures, instead of aspirating it with a pipette. The sEV depletion was performed by ultracentrifugation of foetal bovine serum (Merck-Sigma-Aldrich, Darmstadt, Germany) in an Optima MAX-XP Benchtop Ultracentrifuge with an MLA-55 fixed-angle rotor (Beckman Coulter, Brea, USA) at 120,000 g for 16 h.
3
Synthesis and Characterization of Silica Nanoparticles for Immunological Assays
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FeCl2 · 4H2O and FeCl3 · 6H2O were purchased from Fluka (Buchs, Switzerland). Sodium hypochlorite solution (5 wt.%) was from Bochemie (Bohumín, Czech Republic). Tetramethyl orthosilicate (TMOS), (3-aminopropyl)triethoxysilane (APTES), (3-aminopropyl)dimethylethoxysilane (APDMES), RPMI-1640 medium, phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), L-glutamine, and hydroethidine were purchased from Sigma-Aldrich (Steinheim, Germany). Anti-CD3 monoclonal antibody was purchased from Beckman Coulter and fetal calf serum (FCS) from PAA. Fluorescein-labeled Staphylococcus aureus bacteria and opsonizing reagent were purchased from Molecular Probes (Eugene, OR, USA). Cationic surfactant, cetyltrimethylammonium bromide (CTAB) was purchased from Lachema (Brno, Czech Republic). Ammonium hydroxide solution (25%) and ethanol were purchased from Lach-Ner (Neratovice, Czech Republic). Gentamycin was purchased from Sandoz (Bratislava, Slovakia), cyclophosphamide (Cyf) from Baxter (Deerfield, IL, USA), and [3H]-thymidine from Moravek Biochemicals (Brea, CA, USA). Scintillation fluid was purchased from Perkin Elmer (Waltham, MA, USA). Ultrapure Q-water ultrafiltered on a Milli-Q Gradient A10 system (Millipore, Molsheim, France) was used for preparation of the solutions. ELISA sets were purchased from Affymetrix e-Biosciences.
4
Culturing HT-29 Colon Cancer Cells
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HT-29 human colon adenocarcinoma cells were purchased from LGC STANDARDS (cat. No. ATCC HTB-38) and cultured in a specific pathogen-free cell culture laboratory at 37°C in 5% CO2. HT-29 cells were maintained in McCoy's 5a Medium Modified (Cat No. M8403-500 mL Sigma-Aldrich, St Louis, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Standard Quality; PAA Laboratories GmbH, Pasching, Austria), 160 μg/ml gentamycin (Sandoz, Sandoz GmbH, Austria), and 125 μg/ml amphotericin B (Sigma-Aldrich, St Louis, USA).
5
Culturing Cancer Cell Lines
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Breast (MCF7, T47D) and lung cancer (A549, H1650) cell lines were purchased from ATCC (Manassas, VA, USA), and DSMZ (Braunschweig, Germany), and used for in vitro experiments. MCF7 human breast adenocarcinoma (HTB-22) and H1650 lung adenocarcinoma (CRL-5883) cell lines were cultured in RPMI1640 (LM-R1640; Biosera, Nuaille, France). A549 lung carcinoma (CCL-185) cell line was grown in Dulbecco’s modified Eagle medium (DMEM with 4.5 g/L glucose, BE12-604Q; Lonza, Basel, Switzerland). T47D human ductal carcinoma (HTB-133) cell line was maintained in Ham’s F12 Nutrient Mix (21765-029; Thermo Fisher Scientific, Waltham, MA, USA).
All media were supplemented with 10% fetal bovine serum (FB-1090; Biosera, Nuaille, France) and 0.4% gentamycin (Sandoz, Basel, Switzerland; 80 mg/2 mL). For T47D, 10 µg/mL insulin (12585-014; Thermo Fisher Scientific, Waltham, MA, USA) also was added to medium. All of the cell lines were kept under standard culture conditions (5% CO2, 37 °C).
All cell lines were regularly tested for Mycoplasma sp., applying a PCR test following the methodological article written by Uphoff et al. [65 (link)]. Only negative cell lines were used for research purposes.
All media were supplemented with 10% fetal bovine serum (FB-1090; Biosera, Nuaille, France) and 0.4% gentamycin (Sandoz, Basel, Switzerland; 80 mg/2 mL). For T47D, 10 µg/mL insulin (12585-014; Thermo Fisher Scientific, Waltham, MA, USA) also was added to medium. All of the cell lines were kept under standard culture conditions (5% CO2, 37 °C).
All cell lines were regularly tested for Mycoplasma sp., applying a PCR test following the methodological article written by Uphoff et al. [65 (link)]. Only negative cell lines were used for research purposes.
6
Genomic DNA Isolation from HT29 Colon Cancer Cells
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The HT29 undifferentiated colon adenocarcinoma cell line was purchased from the 1st Department of Pathology and Experimental Cancer Research (Semmelweis University, Budapest, Hungary). The cells were maintained in RPMI 1640 medium (Sigma-Aldrich, United States) supplemented with 10% (v/v) fetal bovine serum (FBS; Standard Quality; PAA Laboratories GmbH, Austria), 125 μg/ml amphotericin B (Sigma-Aldrich, United States), and 160 μg/ml gentamycin (Sandoz, Sandoz GmbH, Austria). The medium was replaced every second day.
Genomic DNA (gDNA; g) was isolated from 5 × 107 steady state, proliferating HT29 cells. DNA isolation was performed by using a High Pure PCR template preparation kit containing proteinase K (Roche GmbH, Germany). The DNA samples were treated with 5 μl RNase A/T1 Mix (Thermo Scientific, Germany). The DNA concentration was determined by Nanodrop (Thermo Scientific, Germany). Gel electrophoresis determined that the fragment length of gDNA was approximately 10,000 base pairs [22 (link)].
According to the bisulfite sequencing analysis of Ogoshi et al. [30 (link)], the basal methylation status of HT29 cells’ CpG sites is as follows: 31.6% in the low range; 11.6 percent in the middle; and 56.7 percent in the high range. Based on MALDI-TOF mass spectrometry measurements, the DNA samples were free of RNA, protein, or lipopolysaccharide contamination (data not shown).
Genomic DNA (gDNA; g) was isolated from 5 × 107 steady state, proliferating HT29 cells. DNA isolation was performed by using a High Pure PCR template preparation kit containing proteinase K (Roche GmbH, Germany). The DNA samples were treated with 5 μl RNase A/T1 Mix (Thermo Scientific, Germany). The DNA concentration was determined by Nanodrop (Thermo Scientific, Germany). Gel electrophoresis determined that the fragment length of gDNA was approximately 10,000 base pairs [22 (link)].
According to the bisulfite sequencing analysis of Ogoshi et al. [30 (link)], the basal methylation status of HT29 cells’ CpG sites is as follows: 31.6% in the low range; 11.6 percent in the middle; and 56.7 percent in the high range. Based on MALDI-TOF mass spectrometry measurements, the DNA samples were free of RNA, protein, or lipopolysaccharide contamination (data not shown).
7
Cultivation of HDFα cells
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HDFα cells were purchased from Life technologies (cat. No. C0135C) and cultured in a specific pathogen-free cell culture laboratory at 37°C in 5% CO2. HDFα cells were maintained in Medium 106 (Life Technologies Corporation, Carlsbad, USA) supplemented with LSGS (Life Technologies Corporation, Carlsbad, USA) and amphotericin B (Sigma). 160 μg/ml gentamycin (Sandoz, Sandoz GmbH, Austria).
8
Isolation and co-culture of CLL cells
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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Histopaque (Sigma-Aldrich, St. Louis, MO, USA). The ratio of the CLL cells among the PBMCs was over 90% assessed by flow cytometry. The isolated CLL cells were cultured at a concentration of 2 × 10 6 cells/ml in RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), gentamycin (Sandoz, Boucherville, QC, Canada), and L-glutamine (Gibco, Carbbad, CA, USA). The CLL cells were cultured in medium alone, on VCAM-1-coated plates or in co-culture with BMSCs. The BMSC cultures, isolated from bone marrow aspirates of normal or ITP patients with no abnormal cells determined by flow cytometry, were prepared as described previously [19] . BMSCs were cultured on 24-well plates maintained at a concentration of 2 × 10 4 cells/ml in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 20% FBS, gentamycin, and L- glutamine. In co-culture experiments after discarding the DMEM, 2 × 10 6 cells/ml CLL cells per well were seeded onto the BMSC monolayer.
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