Human fetal fibroblasts were extracted from fetal skin samples, as described previously in37 (link). Human postnatal dermal fibroblasts and keratinocytes were isolated and expanded from foreskin samples, as described previously21 (link),38 (link). Briefly, skin samples were cut into small pieces and digested overnight at 4 °C in 12 U ml−1 dispase (BD Biosciences, Allschwil, Switzerland) in Hank's balanced salt solution containing 5 μg ml−1 gentamycin (all from Invitrogen, Basel, Switzerland). Subsequently, epidermis was mechanically separated from dermis using forceps. Epidermis was used for the isolation of keratinocytes while dermis for extraction of fibroblasts. For keratinocytes isolation, epidermal pieces were further digested in 0.5% Trypsin–EDTA (Thermo Fisher Scientific, Basel, Switzerland) at 37 °C for 3 min. Keratinocytes were cultivated in serum free keratinocyte medium (CnT-57, CellnTec, Bern, Switzerland) containing 5 μg/ml gentamycin (Thermo Fisher Scientific, Basel, Switzerland). For fibroblasts isolation, dermis was digested in 2 mg ml−1 collagenase blend F (Sigma, Buchs, Switzerland) at 37 °C for ∼ 60 min. Dermal fibroblasts were grown in DMEM supplemented with 10% fetal calf serum (FCS), 4 mM l -alanyl-l -glutamine, 1 mM sodium pyruvate, and 5 μg ml−1 gentamycin (all from Thermo Fisher Scientific, Basel, Switzerland).
Gentamycin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Switzerland, Belgium, France, Canada, Italy, China, Spain, Netherlands, Iceland, India, Austria, Australia, Japan
Gentamycin is a broad-spectrum antibiotic used in laboratory settings. It is effective against a variety of gram-negative and some gram-positive bacteria. Gentamycin functions by inhibiting bacterial protein synthesis, leading to cell death.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (174 mentions)
L glutamine, by Thermo Fisher Scientific (118 mentions)
Penicillin, by Thermo Fisher Scientific (104 mentions)
Streptomycin, by Thermo Fisher Scientific (101 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (85 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (79 mentions)
Glutamax, by Thermo Fisher Scientific (75 mentions)
Fungizone, by Thermo Fisher Scientific (56 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (54 mentions)
Rpmi 1640, by Thermo Fisher Scientific (51 mentions)
Amphotericin b, by Thermo Fisher Scientific (50 mentions)
Hepes, by Thermo Fisher Scientific (39 mentions)
Ciprofloxacin, by Thermo Fisher Scientific (36 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (34 mentions)
Ampicillin, by Thermo Fisher Scientific (33 mentions)
Tetracycline, by Thermo Fisher Scientific (32 mentions)
Fetal bovine serum (fbs), by Merck Group (31 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (29 mentions)
Chloramphenicol, by Thermo Fisher Scientific (27 mentions)
Kanamycin, by Thermo Fisher Scientific (27 mentions)
1 038 protocols using gentamycin
1
Isolation and Culture of Human Skin Cells
2
Cell Culture Conditions for Cell Lines
3
Cell Culture Conditions for Cancer Studies
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HeLa, A-10, SK-OV-3, and SK-OV-3 MDR1-M6/6 cells were grown in Basal Medium Eagle (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Hyclone, GE Life Sciences, Logan, UT, USA) 1% GlutaMAX (Gibco, Life Technologies, Waltham, MA, USA) and 50 µg/mL gentamycin (Life Technologies, USA). HeLa WTβIII cells were grown in Dulbecco’s Modified Eagle Medium (Life Technologies) supplemented with 10% FBS and 50 µg/mL gentamycin. MDA-MB-435 cells were maintained in Improved Minimum Essential Medium (Life Technologies) supplemented with 10% FBS and 25 µg/mL gentamycin. All cells were grown at 37 °C in a humidified environment with 5% CO2.
4
Articular Cartilage Explant Culture
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Articular cartilage was separated from the underlying subchondral bone using a scalpel. Cartilage explants were washed with phosphate‐buffered saline (PBS) containing 50 μg/mL gentamycin (Life Technologies), 0.5 μg/mL amphotericin B (Life Technologies) for 5 minutes. Cartilage explants were kept in Dulbecco's modified eagle medium (DMEM) with 4.5 g/L glucose (Sigma), 25 μg/mL gentamycin (Life technologies), 2 mM Glutamax (Life Technologies), and 10% fetal bovine serum for 24 hours following dissection. The facets were cultured for 96 hours in serum‐free DMEM, 25 μg/mL gentamycin (Life Technologies), 1× Glutamax (Life Technologies), Insulin‐transferrin‐selenium (ITS, Life Technologies), 50 μg/mL ascorbic acid on volume per weight basis (10×, v/w) at 37°C, 5% CO2.
5
Murine Brain Slice Co-Culture Assay
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Animal studies were conducted according to protocols approved by the IACUC of the University of California, Davis. Fresh brain tissue sections from wild-type FvB mice 12–15 weeks of age were prepared under sterile conditions immediately after sacrifice using a precision brain slicer (Braintree Scientific, Inc). High quality 1mm coronal tissue slices were cut in half (sagittal plane) in dissection media (minimum essential medium with glutamine, 1% penicillin/streptomycin, 50μg/mL gentamycin (Thermo), and 4.5mg/mL D-glucose (Sigma)). The matching left and right hemisphere sections were then placed on sterile porous (0.4μm) membranes in a 12-well Transwell plate (Corning) and co-cultured with 250,000 U251 cells either expressing control pMX-GFP or pMX-Nrdp1-FLAG-IRES-GFP in brain culture media (50% dissection media, 25% Hanks’ balanced salt solution, 25% horse serum, 1% penicillin/streptomycin, 50μg/mL gentamycin, 1x non-essential amino acids (Thermo)). Media was replaced in both the upper and lower chambers of the transwell plate after 2–3 days. At 5 days, the tissue was gently washed with PBS, fixed in 10% neutral buffered formalin, and embedded in 1% agarose for stability during histological processing and paraffin-embedding. Representative 5μm thick sections about 50, 100, and 200μm deep were processed for both H&E and anti-GFP (Clontech) IHC.
6
Culturing Procyclic Trypanosoma brucei
7
In vitro culture of murine BV2 and PC12 cells
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Murine microglial BV2 cells (25 (link)) were cultured in RPMI medium supplemented with 5% fetal calf serum, 100 μM non-essential amino acids, 2 mM l-alanyl-glutamine, and 50 μg/ml gentamycin (all ThermoFisher Scientific, Poole, UK) at 37°C in 5% CO2. PC12 cells (ATCC) were cultured in RPMI medium supplemented with 10% normal horse serum, 5% fetal calf serum, 100 μM non-essential amino acids, 2 mM l-alanyl-glutamine, and 50 μg/ml gentamycin (all ThermoFisher Scientific, UK) at 37°C in 5% CO2.
8
Splenocyte Proliferation and Cytokine Assay
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The spleen was processed to a single cell suspension by squeezing through a falcon nylon filter (VWR International, Denmark) and washed three times in RPMI-1640 (Lonza) supplemented with 0.05 mg/mL gentamycin (Thermo Fisher Scientific). Cells were dispersed and counted to a concentration of 3.0×106 cells/mL in RPMI-1640 medium containing 1% Nutridoma (Roche), 1.5 mM monothioglycerol (Sigma), 0.05 mg/mL gentamycin, and 2.5% fetal calf serum (Thermo Fisher Scientific). In total, 5.4×105 and 2.4×105 cells were plated on a flat bottomed 96-well Nunc plate for assessment of proliferation and cytokine production. Cells were stimulated with a final concentration of 5, 25, or 125 µg/mL of OVA and the proliferation plates were incubated for 6 days at 37°C and 5.5% CO2. The cells were treated with 3H-thymidine for the last 17 hours and splenocyte proliferation was measured by counting the incorporated radiolabel on a Wallac Microbeta 1450 liquid scintillation counter (Wallac, Wellesley, USA) after harvesting the cells on a Tomtec-96-well plate harvester (Tomtec Inc, Hamden, CT, USA). For measurement of cytokine levels, the cell concentration in the wells was adjusted to 5.4×105 and supernatants were collected after 5 days of culture.
9
Multilineage Differentiation of ASCs
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For adipogenic, osteogenic, and chondrogenic differentiation, adipose-derived stromal cells (ASCs) (P3, n =3 independent donors) were seeded onto 5 cm cell culture dishes and cultivated in complete α-MEM medium with the addition of recombinant human FGF (50 ng/mL, PeproTech, Hamburg, Germany) until they reached the confluence of 80–90%. Subsequently, the regular culture medium was replaced with respective differentiation media for an additional 2–4 weeks. For adipogenic differentiation, ASCs were cultivated in MesenCultTM adipogenic differentiation medium (StemCell, Cologne, Germany), according to the supplier instructions. For osteogenic differentiation, the cells were cultured with low glucose DMEM (Thermo Fisher Scientific, Basel, Switzerland) supplemented with 0.1 μM dexamethasone, 10 mM glycerol phosphate, 50 μM L-ascorbic acid, 10% FBS, and 50 μg/mL gentamycin (all from Thermo Fisher Scientific, Basel, Switzerland). The chondrogenic differentiation medium consisted of high glucose DMEM (Thermo Fisher Scientific, Basel, Switzerland) supplemented with 50 μg/mL L-ascorbic acid, 40 μg/mL L-proline, 1% ITS™+ Premix (5 μg/mL insulin, 5 μg/mL transferrin, and 5 ng/mL selenious acid), 10 ng/mL TGF-β3, and 50 μg/mL gentamycin.
10
Cell Culture and Dengue Virus Propagation
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A549 (ATCC, CCL-185) and Vero (ATCC, CCL-81) cells were grown in Eagle's minimum essential medium (MEM, GIBCO) supplemented with 10 and 5% fetal bovine serum (GIBCO), respectively, and 50 μg/mL gentamycin (Thermo Fisher Scientific).
HEK-293 cells (ATCC, CRL-1573) were grown in DMEM/F12 (GIBCO) supplemented with 10% fetal bovine serum and 50 μg/mL gentamycin.
C6/36 mosquito cell line from Aedes albopictus, was cultured in L-15 medium (Leibovitz, GIBCO) supplemented with 0.3 % tryptose phosphate broth (Sigma), 0.02% glutamine (Sigma), 1% MEM non-essential amino acids solution (GIBCO) and 10% fetal bovine serum.
DENV strains DENV-1 (Hawaii), DENV-2 (New Guinea C), DENV-3 (H87), and DENV-4 (8,124) were provided by Dr. A.S. Mistchenko (Hospital de Ninos Dr. Ricardo Gutierrez, Buenos Aires, Argentina). Virus stocks were prepared by infecting C6/36 cells with the appropriate DENV serotype at a MOI of 0.1. Supernatants from day 3–7 p.i. were harvested, clarified, filtered, and stored at −80°C until use. Quantification of the virus titers was performed by a plaque assay (see Section 4.4 below). The titer was expressed as the number of plaque-forming units (PFU) per milliliter.
HEK-293 cells (ATCC, CRL-1573) were grown in DMEM/F12 (GIBCO) supplemented with 10% fetal bovine serum and 50 μg/mL gentamycin.
C6/36 mosquito cell line from Aedes albopictus, was cultured in L-15 medium (Leibovitz, GIBCO) supplemented with 0.3 % tryptose phosphate broth (Sigma), 0.02% glutamine (Sigma), 1% MEM non-essential amino acids solution (GIBCO) and 10% fetal bovine serum.
DENV strains DENV-1 (Hawaii), DENV-2 (New Guinea C), DENV-3 (H87), and DENV-4 (8,124) were provided by Dr. A.S. Mistchenko (Hospital de Ninos Dr. Ricardo Gutierrez, Buenos Aires, Argentina). Virus stocks were prepared by infecting C6/36 cells with the appropriate DENV serotype at a MOI of 0.1. Supernatants from day 3–7 p.i. were harvested, clarified, filtered, and stored at −80°C until use. Quantification of the virus titers was performed by a plaque assay (see Section 4.4 below). The titer was expressed as the number of plaque-forming units (PFU) per milliliter.
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