H727, H460, H82, SW2, and H510 cells cultured in RPMI-1640 (Sigma-Aldrich), containing 10% FCS, 50 µg/ml gentamycin, 2,5 mM L-glutamine, A549 in DMEM (Sigma-Aldrich), containing 10 % FCS, 50 µg/ml gentamycin, 2,5 mM L-glutamine. All cell lines were originally from ATCC and were received from Nycomed. They were tested for mycoplasma contamination prior to experiments. For GC bioassay and T cell activation assay cells were cultured for 24 h in phenol red-free, 5% steroid-free FCS containing DMEM (50 µg/ml gentamycin, 2,5 mM L-glutamine). The supernatant was collected and boiled for 15 min at 90 °C.
Gentamycin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Australia, Italy, France, Switzerland, Canada, Austria, Sao Tome and Principe, India, Netherlands, Hungary, Poland, Macao
Gentamycin is a laboratory reagent used for the detection and quantification of the antibiotic gentamicin in various samples. It is a widely used tool in pharmacological and medical research applications.
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Lab products found in correlation
Streptomycin, by Merck Group (60 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (48 mentions)
Fetal bovine serum (fbs), by Merck Group (48 mentions)
L glutamine, by Merck Group (48 mentions)
Penicillin, by Merck Group (47 mentions)
Penicillin streptomycin, by Merck Group (31 mentions)
Amphotericin b, by Merck Group (30 mentions)
L glutamine, by Thermo Fisher Scientific (30 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (26 mentions)
Kanamycin, by Merck Group (24 mentions)
Glutamax, by Thermo Fisher Scientific (23 mentions)
Ampicillin, by Merck Group (23 mentions)
Bovine serum albumin (bsa), by Merck Group (21 mentions)
Chloramphenicol, by Merck Group (20 mentions)
Insulin, by Merck Group (20 mentions)
Gentamycin, by Thermo Fisher Scientific (19 mentions)
Erythromycin, by Merck Group (19 mentions)
Vancomycin, by Merck Group (18 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (18 mentions)
Rpmi 1640, by Merck Group (17 mentions)
596 protocols using gentamycin
1
Cell Culture Conditions for Bioassays
2
Maintaining Divergens and Falciparum Parasite Cultures
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The Rouen 1987 strain of B. divergens was kindly provided by Dr. Stephane Delbecq (Laboratoire de Biologie Cellulaire et Moleculaire UFR Pharmacie, Montpellier, France). Asynchronous B. divergens parasite cultures were maintained in human erythrocytes (type O+) suspended in complete culture medium [RPMI-1640 medium supplemented with 25.2 mM HEPES, 22.2 mM D-glucose, 50 mg/l hypoxanthine, 21.4 mM sodium bicarbonate, 48 mg/l gentamycin (Sigma)] further supplemented with 10% human serum [33 (link)]. Cultures were maintained at 37°C in a gaseous environment of 90% N2, 5% O2 and 5% CO2 on a rotary platform (60 rpm) at ~5% hematocrit and 10–15% parasitemia, with daily media replacement. Comparatively, intra-erythrocytic P. falciparum (3D7 strain) parasites were obtained from the MR4 (www.mr4.org ) and were maintained at a 5% hematocrit, 2–5% parasitemia in complete culture medium [RPMI-1640 medium supplemented with 25.2 mM HEPES, 22.2 mM D-glucose, 50 mg/l hypoxanthine, 21.4 mM sodium bicarbonate, 48 mg/l gentamycin (Sigma)] further supplemented with 0.5% Albumax II (Invitrogen) [34 (link)] in the same gaseous environment under shaking conditions as above.
3
Isolation of FDB Muscle Fibers
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FDB fibers were isolated according to procedures described by Schuh et al. (2012 (link)). Following surgical excision of the FDB from both feet, the muscles were incubated in 1 mL of pre-warmed dissociation media (DMEM, Gibco 10566016; 4.5 mg/ml glucose, 2% FBS, Bovogen Biologicals; 4 mg/mL collagenase A, Roche 10103586001; 50 μg/mL gentamycin, Sigma Aldrich, Australia G1397) for 1 h 45 min (37°C, 5% CO2). Following the incubation period, FDB muscles were removed from collagenase And placed into ~1.5 mL of incubation media [DMEM containing 4.5 mg/ml glucose, no phenol red (Gibco), 2.0% FBS, 0.1% gentamycin solution (Sigma Aldrich, Australia G1397)] and triturated with pipette tips of decreasing bore size to yield isolated fibers. Fibers were then plated according to analysis methods described below.
4
Bovine Cartilage Chondrocyte Isolation
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Bovine cartilage was harvested from the lower leg joints of a young calf. Briefly, the ankle joint was cut open along the joint line and cartilage tissue was cut with a scalpel into thin sections parallel to the subchondral bone. Chondrocytes were isolated by digesting in Dulbecco’s modified Eagle’s medium (DMEM) + Glutamax (4.5 g l−1 glucose) with 0.2% w/v pronase, 10 mM HEPES and 50 μg ml−1 gentamycin (all reagents from Invitrogen, Paisley, UK) for 1 h at 37 °C with agitation. This digest was removed and replaced with DMEM + Glutamax (4.5 g l−1 glucose) supplemented with 10 mM HEPES, 50 μg ml−1 gentamycin, 5% v/v fetal bovine serum (FBS) and 0.04% w/v collagenase (Sigma) overnight at 37 °C with agitation. After digestion, isolated chondrocytes were filtered through a 70 μm pore size filter, centrifuged at 250 x g for 3 min, and plated in DMEM (4.5 g l−1 glucose) with 10% v/v FBS, 50 μg ml−1 ascorbic acid (Sigma) and 50 μg ml−1 gentamycin (expansion medium).
5
Visualization of E. coli Infection in T84 Cells
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T84 cells were seeded on coverslips in 24-well plates and infected with E coli–LF82 or E coli–HB101 (4 h, 108 CFU/mL), fixed with 4% paraformaldehyde for 10 minutes, washed, and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (Sigma, 2 × 5 min.) and blocked with 10% donkey serum (room temperature, 30 min). After addition of anti–E coli antibody (1:50, 30 min, 37ºC; Abcam, Cambridge, UK), washing (2 × 5 min), donkey anti-goat secondary Alexa Fluor–488 antibody (1:1000, 30 min, room temperature), cells were washed (2 × 5 min), stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000, 5 min, room temperature), rinsed, mounted with Dako fluorescent mounting medium (Dako North America, Inc, Agilent Technologies, CA), and viewed on an Olympus (Tokyo, Japan) BX41 wide-field fluorescence microscope.
The gentamycin assay assessed bacterial internalization.15 (link) T84 cells were grown to 50%–70% confluence as determined by phase-contrast microscopy, infected with E coli, a sample of the medium was collected, and then gentamycin (100 μg/mL, 1 h; Sigma) was added to the culture wells. After rinsing, epithelia were lysed with 1% Triton X-100, and the lysate was cultured for 18 hours at 37ºC on blood agar plates: CFUs were counted and data are presented as the percentage of internalization.
The gentamycin assay assessed bacterial internalization.15 (link) T84 cells were grown to 50%–70% confluence as determined by phase-contrast microscopy, infected with E coli, a sample of the medium was collected, and then gentamycin (100 μg/mL, 1 h; Sigma) was added to the culture wells. After rinsing, epithelia were lysed with 1% Triton X-100, and the lysate was cultured for 18 hours at 37ºC on blood agar plates: CFUs were counted and data are presented as the percentage of internalization.
6
Recombinant Salmonella Infection of Tumor Cells
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Infection of tumor cells with recombinant Salmonella strains was conducted as described by Vendrell et al. (53 (link)). Briefly, Ramos cells were cultured in 48-well plates (500,000 cells/well) in 1 mL of Advanced RPMI 1640 medium supplemented with 2% FBS without antibiotic. The cells were infected at a Multiplicity of Infection (MOI) of 100 with previously induced recombinant bacteria. After centrifugation at 2,000 rpm for 5 min to foster the interaction between bacteria and cells, they were incubated at 37°C and 5% CO2 for 2–10 h. Subsequently they were washed twice in base advanced RPMI 1640 medium supplemented with gentamycin (Sigma-Aldrich) 100 μg/mL and finally resuspended in 1 mL of advanced RPMI 1640 medium supplemented with 2% FBS and 50 μg/mL gentamycin and used in the cellular viability and apoptosis detection protocols. Vincristine (Sigma-Aldrich) 0.5 nM diluted in injectable water was used as a positive control in all cases; this drug is used as treatment for NHL.
7
Skeletal Muscle Cell Differentiation and WNT Signaling
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C25, 16U, 54-6 cells were cultured in Promocell skeletal muscle growth medium (Promocell, C-23060) supplemented with 15% foetal calf serum (FBS) and 1:1000 Gentamycin (Sigma). Differentiation was induced by switching the medium to DMEM GlutaMax, 0.5% FBS, 1:1000 bovine Insulin (Sigma) and 1:1000 Gentamycin. RH30 were cultured in DMEM GlutaMax (Gibco, 10566016) supplemented with 10% FBS and 1% Pen/Strep (Sigma). Cells were maintained in a humidified incubator at 37 °C and 5% CO2. Differentiation medium was used for RH30 cell culture under low serum conditions.
For WNT ligand stimulation experiments, cells were plated in 6 well plates and 24 h later were stimulated with 10 ng/µl WNT3a or WNT7a (final concentration) and protein harvested 24 h later.
For WNT ligand stimulation experiments, cells were plated in 6 well plates and 24 h later were stimulated with 10 ng/µl WNT3a or WNT7a (final concentration) and protein harvested 24 h later.
8
Hippocampal Primary Neuron Culture Protocol
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Hippocampal primary cultures were prepared as previously described (33 (link)). Briefly, hippocampi isolated postnatal day 0 to 3 Wistar rats were digested by three separate enzymatic [trypsin (5 mg/ml; Sigma-Aldrich, no. T10051), deoxyribonuclease type I (0.75 mg/ml; Sigma-Aldrich, no. D5025-150KU), trypsin inhibitor (2.5 mg/ml; Sigma-Aldrich, no. T9003)] and mechanic steps alternated with washings [4.2 mM NaHCO3, 12 mM Hepes (Merck, no. 4034), 200 μM kynurenic acid (Sigma-Aldrich, no. K3375), 25 μM D-AP5 (Abcam, no. ab120003), powder Hank’s modified (Sigma-Aldrich, no. H-2387), 33 mM glucose, bovine serum albumin (BSA) fraction V (0.3 mg/ml; Merck, no. 10735086001), 12 mM MgSO4, and gentamycin (0.25 μl/ml; Sigma-Aldrich, no. G12)]. The final pellet of cell was resuspended in Neurobasal medium [1× Neurobasal-A Medium (Gibco, no. 10888-022) supplemented with 1× GlutaMAX (Gibco, no. 35050-061), 1× B27 supplement (Life Technologies, no. 17504044), and gentamycin (0.5 μl/ml; Sigma-Aldrich, no. G1272)]. Cells were then plated on a 12 mm–by–24 mm coverslip (150,000 cells per coverslip) pretreated with poly-l -ornithine (Sigma-Aldrich, no. P4957) and maintained (7 to 10 days in vitro) in an incubator at 37°C and 5% CO2.
9
Cell Culture Conditions for Diverse Cell Lines
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The non-polarized cell lines HeLa, INT407 and polarized cell lines Caco-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), high-glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; GIBCO) and 50 μg/ml gentamycin (Sigma-Aldrich). The culture medium was changed every 4 days in Caco-2 cells. The culture medium was changed every 4 days. Polarized cell lines T-84 cells were cultured in Dulbecco's modified Eagle's medium nutrient mixture F-12 HAM (DMEM/F-12, 1:1; Sigma-Aldrich) supplemented with 10% FBS and 50 μg/ml gentamycin. The culture medium was changed every 2 days. All cells were incubated in 37°C in a humidified atmosphere containing 5% CO2.
10
Cell Culture Protocols for Tumor and Immune Cell Lines
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