All cells were grown in a humidified atmosphere containing 5% CO2 at 37°C. HeLaP4 310 LEDGF/p75 depleted cells ([46 ], further referred to as LEDGFKD cells) were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Merelbeke, Belgium) supplemented with 5% v/v heat inactivated fetal calf serum (FCS; Sigma-Aldrich, Bornem, Belgium), 0.005% w/v gentamicin (GIBCO), 0.05% w/v geneticin (GIBCO) and 0.01% w/v zeocin (Life Technologies, Ghent, Belgium). These cells are monoclonal LEDGFKD cells, derived from HeLaP4 cells (gift from P. Charneau, Institut Pasteur, Paris, France). HelaP4 cells were grown on DMEM (GIBCO) supplemented with 5% v/v heat inactivated fetal calf serum (FCS; Sigma-Aldrich), 0.005% w/v gentamicin (GIBCO) & 0.05% w/v geneticin (GIBCO). HEK 293T cells (gift from O. Danos, Evry, France) were cultured in DMEM medium (GIBCO) with 8% v/v heat inactivated FCS (Sigma-Aldrich) and 0.005% w/v gentamicin (GIBCO). SupT1 cells were cultured in Roswell Park Memorial Institutes medium (RPMI, GIBCO-BRL, Merelbeke, Belgium) supplemented with 10% v/v heat inactivated fetal calf serum FCS (Sigma-Aldrich, Bornem,Belgium) and 0.005% w/v gentamicin (GIBCO). Nalm pre-B cells were cultured in RPMI (GIBCO) with 10% v/v heat inactivated FCS (Sigma-Aldrich) and 0.005% w/v gentamicin (GIBCO).
Gentamicin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Canada, France, Belgium, Switzerland, Italy, Spain, China, Ireland, Israel, Sweden, Austria, Australia, Japan, India, Argentina, Denmark, Netherlands, Macao, Brazil, Portugal, Panama
Gentamicin is a laboratory reagent used for the detection and quantification of the antibiotic gentamicin in biological samples. It is a commonly used tool in research and clinical settings.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (504 mentions)
L glutamine, by Thermo Fisher Scientific (271 mentions)
Penicillin, by Thermo Fisher Scientific (263 mentions)
Streptomycin, by Thermo Fisher Scientific (251 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (210 mentions)
Glutamax, by Thermo Fisher Scientific (209 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (203 mentions)
Ciprofloxacin, by Thermo Fisher Scientific (178 mentions)
Hepes, by Thermo Fisher Scientific (138 mentions)
Amphotericin b, by Thermo Fisher Scientific (135 mentions)
Fungizone, by Thermo Fisher Scientific (126 mentions)
Tetracycline, by Thermo Fisher Scientific (115 mentions)
Rpmi 1640, by Thermo Fisher Scientific (110 mentions)
Ampicillin, by Thermo Fisher Scientific (108 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (105 mentions)
Chloramphenicol, by Thermo Fisher Scientific (89 mentions)
Fetal bovine serum (fbs), by Merck Group (89 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (89 mentions)
Erythromycin, by Thermo Fisher Scientific (87 mentions)
Amikacin, by Thermo Fisher Scientific (87 mentions)
2 480 protocols using gentamicin
1
Cell Culture Protocols for LEDGFKD and Control Cells
2
Cultivation of Parasites and Host Cells
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Type I RH parasites (ATCC PRA-319) were grown in human foreskin fibroblasts (HFFs, ATCC SCRC-1041) and maintained in DMEM (GIBCO) supplemented with 2 mM glutamine, 10% inactivated fetal bovine serum (IFS), and 10 μg/ml gentamicin. C57Bl/6J mouse embryonic fibroblasts (B6 MEFs) and RAW 264.7 macrophages were maintained in DMEM (GIBCO) supplemented with 2 mM glutamine, 10% IFS, and 10 μg/ml gentamicin (Thermo Fisher Scientific). When indicated, Human Plasma-like Media (HPLM, GIBCO) was used as base media and supplemented with 2 mM glutamine, 10% IFS, and 10 μg/ml gentamicin.
The NF54 P. falciparum line was cultured and synchronized as described previously163 (link),164 (link). Cultures were kept at 37°C in RPMI-1640 medium supplemented with 25 mM HEPES, 100 μM hypoxanthine, 0.5% Albumax II, 24 mM sodium bicarbonate, 25 μg/mL gentamicin (Life Technologies, Carlsbad, CA 11021–045) and gassed with 5% CO2, 1% O2, and 94% N2 mixture.
The NF54 P. falciparum line was cultured and synchronized as described previously163 (link),164 (link). Cultures were kept at 37°C in RPMI-1640 medium supplemented with 25 mM HEPES, 100 μM hypoxanthine, 0.5% Albumax II, 24 mM sodium bicarbonate, 25 μg/mL gentamicin (Life Technologies, Carlsbad, CA 11021–045) and gassed with 5% CO2, 1% O2, and 94% N2 mixture.
3
Generation of Immortalized Mouse OECs
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Immortalized mouse OECs were generated from olfactory bulb ensheathing glia of GFP-expressing mice (C57BL/6-Tg (ACTB-EGFP)1Osb/J, Jackson Laboratory, Bar Harbor, USA [7 (link), 8 (link)]. mOEC-GFP were cultured in Dulbecco’s Modified Eagle Medium/Nutrient F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS, Bovogen) and 10 ng/mL gentamicin (Life Technologies). Dorsal root ganglion Schwann cells and astrocytes were isolated from S100β-DsRed transgenic mice [9 (link)]. Schwann cells and astrocytes were maintained in complete medium conditions (Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 1% GlutaMAX™ (Life Technologies) supplement and 50 ng/mL gentamicin). Pancreatic cancer cell (BxPC-3) and Neural stem cells (ReNcell VM) were maintained in DMEM supplemented with 10% FBS and 10 ng/mL gentamicin and in DMEM/F12 supplemented with 10% fetal bovine serum, 1% N-2 Supplement (Life Technologies) and 10 ng/mL gentamicin, respectively. All the cells were maintained in a humidified incubator at 37 °C and 5% CO2.
4
Culturing Synchronized P. falciparum Parasites
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P. falciparum parasites were cultured in human erythrocytes50 (link) and were synchronised by sorbitol treatment51 (link). NF54-based lines were cultured at 2% haematocrit in human O + erythrocytes (purchased from pooled, anonymous donors at the New York Blood Center) in RPMI 1640 medium (KD Medical) supplemented with 0.21% sodium bicarbonate (Sigma Aldrich), 10 μg/mL gentamicin (Fisher) and 10% O + human serum. Dd2-B2-PfATP4G358S and Dd2-B2 parasites were cultured at 3% haematocrit in human O + erythrocytes in RPMI-1640 media, supplemented with 25 mM HEPES (Fisher), 50 mg/L hypoxanthine (Sigma Aldrich), 2 mM l -glutamine (Cambridge Isotope Laboratories, Inc.), 0.21% sodium bicarbonate (Sigma Aldrich), 0.5% (wt/vol) AlbuMAXII (Invitrogen), 7.5% O + human serum and 10 μg/mL gentamicin (Fisher). Cultures were maintained at 37 °C in an atmosphere consisting of 5% O2, 5% CO2 and 90% N2.
For all other parasites, the cultures, which typically had a haematocrit between 2-4%, were maintained with continuous shaking52 (link) at 37 °C in a low-O2 atmosphere (1% O2, 3% CO2 and 96% N2). The culture medium was RPMI 1640 containing 25 mM HEPES (Gibco) supplemented with 11 mM additional glucose, 0.2 mM hypoxanthine, 20 μg/mL gentamicin sulphate and 3 g/L Albumax II. The blood was provided by Australian Red Cross Lifeblood without disclosing the identities of the donors.
For all other parasites, the cultures, which typically had a haematocrit between 2-4%, were maintained with continuous shaking52 (link) at 37 °C in a low-O2 atmosphere (1% O2, 3% CO2 and 96% N2). The culture medium was RPMI 1640 containing 25 mM HEPES (Gibco) supplemented with 11 mM additional glucose, 0.2 mM hypoxanthine, 20 μg/mL gentamicin sulphate and 3 g/L Albumax II. The blood was provided by Australian Red Cross Lifeblood without disclosing the identities of the donors.
5
Cell Culture Conditions for Various Cell Lines
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Human foreskin fibroblasts (HFF) were cultured in MEM supplemented with GlutaMAX (Life Technologies), 5% fetal bovine serum, 0.5 ng/ml basic fibroblast growth factor (bFGF) and 100 μg/ml gentamicin (Life Technologies). HEC-LTT cells originate from human umbilical vein endothelial cells that contain doxycycline-inducible expression cassettes for SV40 large T antigen and the human telomerase catalytic subunit [23 (link)], and were maintained in endothelial growth medium (EGM BulletKit, Lonza) with 2 μg/ml doxycycline (Sigma-Aldrich) culture vessels coated with 0.1% gelatin for 30 min (Sigma-Aldrich). Infection was performed 1 d after doxycycline withdrawal [24 (link)]. MV9G cells [22 (link)] were cultured in minimum essential medium (MEM) supplemented with GlutaMAX (Life Technologies), 10% fetal bovine serum (PAA), 50 μg/ml G-418 (GE Healthcare) and 100 μg/ml gentamicin (Life Technologies) as previously described [25 (link)]. The human melanoma cell line MeWo [26 (link)–28 (link)] (ATCC-HTB-65) was purchased from LGC Standards. MeWo and Vero cells [29 (link)] (obtained from ATCC) were cultured in Dulbecco's Modified Eagle's medium supplemented with GlutaMAX (Life Technologies), 10% fetal bovine serum (PAA) and 100 μg/ml gentamicin (Life Technologies).
6
Cell Culture Conditions for COVID-19 Research
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Madin-Darby Canine Kidney epithelial (MDCK, American Type Culture collection, ATCC CCL-34), human embryonic kidney (293T; ATCC CRL-11268), human lung epithelial carcinoma (A549; ATCC CCL-185), and African green monkey kidney epithelial E6 (Vero E6, kindly provided by Prof. Luis Enjuanes, Centro Nacional de Biotecnología, CNB-CSIC, Spain) cells were grown at 37°C in air enriched with 5% CO2 using Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco), and 50 μg/ml gentamicin (Gibco). A549 cells overexpressing human ACE-2 (hACE-2, A549-ACE-2), generated in our laboratory, were grown in the same media containing 2.5 μg/ml of blasticidin (ThermoFisher Scientific). Human HAP-1 near-haploid wild-type (WT) cells, derived from a human leukemia, and HAP-1 cells knock-out (KO) for IFI6 using the CRISPR/Cas9 technology were obtained from Horizon Discovery, Inc. These cells were grown at 37°C in air enriched with 5% CO2 using Iscove´s Modified Dulbecco´s medium supplemented with 10% fetal bovine serum (Gibco), and 50 μg/ml gentamicin (Gibco). The non-tumorigenic human bronchial epithelial cell line BEAS-2B (CRL-9609), was obtained from the ATCC and grown at 37°C in air enriched with 5% CO using RPMI medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), and 50 μg/ml gentamicin (Gibco).
7
In vitro maturation of buffalo oocytes
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Unless otherwise stated, all chemicals were purchased from Sigma Argentina. Recovered cumulus-oocyte complexes (COCs) were washed and maintained in TCM 199 supplemented with Hank's salts, 10% v/v FBS (Gibco, Thermo Fisher Scientific, Argentina) and 0.1% v/v gentamicin (Gibco, Thermo Fisher Scientific, gentamicin reagent solution, 50 mg/ml) for classification according to quality. Cumulus-oocyte complex quality was classified according to IETS guidelines (quality I-highest to quality IV-poorest) and previous reports for buffalo (Di Francesco et al., 2011) based on the number of layers of compact cumulus investments and presence of homogenous cytoplasm. Once classified, the COCs were washed and transferred to a 35-mm Petri dish containing 3 ml of maturation medium consisting of TCM 199 with Earl's salts and 25 mM Hepes, 10% v/v FBS, 50 mM cysteamine, 5 μg/ml FSH (NIH-FSH-P1; Folltropin-V; Bioniche Animal Health, Belleville, Ontario, Canada) and 0.1% v/v gentamycin sulfate (Gibco, Thermo Fisher Scientific, gentamicin reagent solution, 50 mg/ml). They were then transferred to 1.8 ml Eppendorf tubes filled to the top with pre-equilibrated maturation medium and allowed to mature in transit to the IVF laboratory (8 h from farm) in a portable incubator (Minitube, Germany) set at 37 °C.
8
Skin Cell Isolation and Cultivation
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Skin cell isolation and the following cultivation was performed as previously described in Böttcher-Haberzeth et al. (Böttcher-Haberzeth et al., 2013 (link)). Briefly, skin samples were reduced to small pieces (about 3 mm3) and digested in 12 U/mL dispase (BD Biosciences, Switzerland) combined with Hank’s balanced salt solution containing 5 mg/ml gentamicin (all from Invitrogen, Switzerland) at 4°C overnight. Thereafter, forceps where used to separate the epidermis and dermis for subsequent cell isolation. Epidermal cells were extracted from the epidermis by further digestion in 1% trypsin and 5 mM EDTA (both from Invitrogen, Switzerland) for 10 min at 37°C and afterwards cultured in serum-free keratinocyte medium containing 25 mg/ml bovine pituitary extract, 0.2 ng/ml epidermal growth factor, and 5 mg/ml gentamicin (all from Invitrogen, Switzerland). Further digestion of the dermal tissue in 2 mg/ml collagenase blend F (Sigma, Switzerland) for 4 h at 37°C maintained to the extraction of fibroblasts, which were further cultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 4 mM L-alanyl-l - glutamine, 1 mM sodium pyruvate, and 5 mg/ml gentamicin (all from Invitrogen, Switzerland).
9
Neuro2A Cell Differentiation
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Neuro2A cells were plated in 24-well plates at a density of 50,000 cells/well in Dulbecco’s modified Eagle medium (DMEM)-Glutamax™ (Thermo Fisher Scientific) supplemented with 6% FBS and 50 µg/mL gentamicin (Invitrogen) for one night to acquire an approximate density of 100,000 cells/well the next day. They were also kept at 37 °C and 5% CO2. In the morning, the medium was replaced by a differentiation medium (DMEM-Glutamax™, 50 µg/mL gentamicin, 0.1% BSA (Acros Organics, Illkirch, France) to allow the formation of neurites and renewed after 24 h.
10
Directed Differentiation of hPSCs
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To observe the effects of the two (2D and 3D) culture systems on differentiation, neither Wnt pathway activators nor inhibitors were added to the media as conventionally done (See Table 1 ). The differentiation of the hPSCs took place in 12-well plates (Nunc) treated with MG for the 2D and in ultra-low attachment non-treated plates for 3D. Mesodermal differentiation proceeded in Stempro 34 (Sigma-Aldrich) medium supplemented with 10× ITS (Gibco), 40 ng/mL BMP4, 10ng/mL FGF2 (Preprotech), 50 μg/mL Ascorbic Acid (Sigma-Aldrich), 100× Glutamax (Gibco) and Gentamicin (Gibco) from day 0 to day 4. On day 4, the medium was changed to Stempro 34 with 10× ITS, 100× Glutamax, Gentamicin, 10 ng/mL FGF, 10 ng/mL VEGF; 10 ng/mL of IL6 and 2 U/mL EPO (Preprotech) for three more days (days 4 to 6). On day 6 the medium was replaced with hematopoietic differentiation Stempro 34 medium containing the above supplements plus 50 ng/mL SCF, 5 ng/mL IL7, 5 ng/mL Flt3 and 10 ng/mL IL3 (Preprotech Rocky Hill, USA). CHIR99021 (3 μM) and IWR-1(1 µM) (Sigma), were added only when indicated.
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