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Gentamicin

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Gentamicin is a broad-spectrum antibiotic commonly used in cell culture media. It is effective against a wide range of Gram-negative and some Gram-positive bacteria. Gentamicin functions by inhibiting bacterial protein synthesis, thereby preventing the growth and replication of susceptible microorganisms.

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Lab products found in correlation

Insulin, by Merck Group (5 mentions) Penicillin, by Merck Group (5 mentions) Penicillin g, by Merck Group (3 mentions) Isoproterenol hydrochloride, by Merck Group (3 mentions) Adenine, by Merck Group (3 mentions) Epidermal growth factor (egf), by Merck Group (2 mentions) Hydrocortisone, by Merck Group (2 mentions) Amphotericin b, by Gemini Bio (2 mentions) Vero 76 cells, by American Type Culture Collection (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Ham s f12, by Thermo Fisher Scientific (2 mentions) Fungizone, by Bristol-Myers Squibb (2 mentions) Cell strainer of 100 m, by Thermo Fisher Scientific (2 mentions) Heparin, by Merck Group (1 mentions) Ultra low attachment 96 well plate, by Corning (1 mentions) Fungizone, by Thermo Fisher Scientific (1 mentions) Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions) Celigo imaging cytometer, by Revvity (1 mentions) Ultra low attachment 6 well plate, by Corning (1 mentions) Fetal bovine serum (fbs), by Gemini Bio (1 mentions)

24 protocols using gentamicin

1

Fibroblast and Keratinocyte Cell Culture with EPA

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Fibroblasts were cultured in the DMEM with a supplementation of 10% bovine growth serum (FB Essence; Seradigm, Mississauga, ON, Canada), 100 Ul/ml penicillin G (Sigma, Oakville, ON, Canada), and 25 μg/ml gentamicin (Gemini Bio-Products, Sacramento, CA). Keratinocytes were cultured in a combination of DMEM with Ham’s F12 (3:1), supplemented with 5% FetalClone II serum (Galenova, Saint-Hyacinthe, QC, Canada), 5 μg/ml insulin (Sigma, Oakville, ON, Canada), 0.4 μg/ml hydrocortisone (Galenova, St-Hyacinthe, QC, CA), 10-10 M cholera toxin (Sigma, Oakville, ON, Canada), 10 ng/ml human epidermal growth factor (Ango Inc, San Ramon, CA), 100 Ul/ml penicillin G (Sigma, Oakville, ON, Canada), and 25 μg/ml gentamicin (Gemini Bio-Products, Sacramento, CA). EPA supplementation was provided by dissolving EPA (Cedarlane, Burlington, ON, Canada) in ethanol in order to generate a stock solution. The EPA solution was then added to the serum to provide a concentration of 10 μM EPA, after evaporation of the remaining ethanol (32 , 33 (link)).
Morin S., Bélanger S., Cortez Ghio S, & Pouliot R. (2023). Eicosapentaenoic acid reduces the proportion of IL-17A–producing T cells in a 3D psoriatic skin model. Journal of Lipid Research, 64(9), 100428.
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2

Isolation and Culture of Human Keratinocytes and Fibroblasts

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Human keratinocytes and fibroblasts were isolated from the foreskin of a healthy individual (14 days old) as previously described [27 (link)]. Primary fibroblasts and HeLa cells (ATCC CCL-2) were cultured in fibroblast medium (Dulbecco-Vogt modified Eagle medium (Gibco™, Waltham, MA, USA) supplemented with 10% Avantor Seradigm FB Essence serum (Avantor®, Radnor, PA, USA), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 25 μg/mL gentamicin (Gemini Bio, West Sacramento, CA, USA)). Human keratinocytes were cultured on a feeder layer composed of irradiated human fibroblasts cultured in keratinocyte medium (Dulbecco-Vogt modified Eagle medium (Gibco™): Ham’s F12 (Gibco™), ratio 3:1, supplemented with 24.25 μg/mL adenine (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 0.4 μg/mL hydrocortisone (Galenova, Saint-Hyacinthe, QC, Canada), 0.212 μg/mL isoproterenol hydrochloride (Sigma-Aldrich), 5% bovine HyClone FetalClone II serum (GE Healthcare, Chicago, IL, USA), 10 ng/mL human epidermal growth factor (Austral Biologicals, San Ramon, CA, USA), 100 U/mL penicillin (Sigma-Aldrich) and 25 μg/mL gentamicin (Gemini Bio)).
Barbier M.A., Ferland K., De Koninck H., Doucet E.J., Dubourget L., Kim M., Cattier B., Morissette A., Bchetnia M., Larouche D., Kim D.H., St-Jean G, & Germain L. (2024). Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo. International Journal of Molecular Sciences, 25(3), 1513.
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3

Culture and Maintenance of KSHV-Infected BJAB Cells

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BJAB cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Gibco, Life Technologies) containing 10% bovine growth serum (BGS) (HyClone GE Healthcare) and 15 μg/ml gentamicin (Gemini bio-product, California, USA). KSHV-infected BJAB cells (Chen and Lagunoff 2005 (link)) were maintained in RPMI medium containing 10% BGS, 15 μg/ml gentamicin, and 17 μg/ml puromycin (InvivoGen, San Diego, California). BJAB cells expressing green fluorescent protein (GFP) from the pEGFP-C1 plasmid, were cultured in RPMI medium containing 10% BGS, 15 μg/ml gentamicin and 800 μg/ml G418 (Gemini bio-product). Three low electrically conductive DEP Buffers were prepared. DEP Buffer I was composed of 8.5% Sucrose (w/v), 0.3% Glucose (w/v), and 0.725% RPMI in DI water. DEP buffer II was composed of 0.0025 g/ml glucose, 0.09 g/ml sucrose, 6 ml of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, life technologies), and 40 ml of DI water. Lastly, DEP buffer III was composed of 135 mg Sucrose, 326mg Dextrose (Sigma Aldrich, MO, USA), and 45 ml of DI water. The conductivity of DEP buffers I, II and III were 100 μS/cm, 1.6 μS/cm and 1μS/cm, respectively. For KSHV-infected BJAB cell lines, we infected BJAB with KSHV recombinant virus carrying an expression cassette for GFP(Chen and Lagunoff 2005 (link)).
Safavieh M., Khetani S., Juillard F., Kaul V., Kanakasabapathy M.K., Kaye K.M, & Shafiee H. (2016). Electrical response of B cell lines latently infected with Kaposi’s sarcoma herpesvirus. Biosensors & bioelectronics, 80, 230-236.
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4

Planarian Stem Cell Ablation Protocol

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Planarians from the asexual strain CIW4 [53 (link)] were kept in 1x Montjuïc water (1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl and 1.2 mM NaHCO3 in Milli-Q water, pH 6.9–8.1) supplemented with 50 μg/mL gentamicin (Gemini Bio-Products # 400–108) [54 (link)]. Worms were kept in unsealed Ziploc containers or 100mm Petri dishes. Worms were kept in unsealed Ziploc containers or 100mm Petri dishes. We irradiated worms on the top shelf of a benchtop X-ray irradiator (CellRad, Precision X-ray) with 60 Gray at 130 kV, 5 mA to ablate stem cells.
, & Bar Yaacov D. (2022). Functional analysis of ADARs in planarians supports a bilaterian ancestral role in suppressing double-stranded RNA-response. PLoS Pathogens, 18(1), e1010250.
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5

Isolation and Culturing of Skin Fibroblasts

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A skin biopsy was performed on the patient who reported cutaneous involvement. The fibroblast cells were isolated from skin biopsies and kept in liquid nitrogen until used as previously described.15 (link) Briefly, fibroblasts were cultivated in Dulbecco's Modified Eagle Medium with 10% fetal bovin serum (Seradigm) with 100 IU/mL penicillin G (Sigma-Aldrich) and 25 μg/mL gentamicin (Gemini Bio-Products) in 8% CO2 at 37°C. They were cultured in flasks until confluency (100%) prior to be split and seeded in a 6-well plate containing sterile glass coverslips.
Beaudin M., Sellami L., Martel C., Touzel-Deschênes L., Houle G., Martineau L., Lacroix K., Lavallée A., Chrestian N., Rouleau G.A., Gros-Louis F., Laforce R J.r, & Dupré N. (2020). Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34. Neurology: Genetics, 6(2), e403.
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6

Tumorsphere Culture and Drug Treatment

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For the tumorsphere assays, cultured cells were plated at a density of 20,000 cells per well in ultra-low attachment 6 well plates (Corning®, USA) in serum-free medium. The serum-free medium consisted of Ham’s F-12 medium supplemented with 2% B27 (Thermo Fisher Scientific, USA), 25 μg/ml gentamicin (Gemini Bioproducts), 2.5 μg/ml fungizone (Thermo Fisher Scientific), 20 ng/ml EGF (Sigma-Aldrich, USA), 20 ng/ml bFGF (Sigma-Aldrich), 4 μg/ml heparin and 0.5 μg/ml hydrocortisone (Sigma-Aldrich, USA). During primary culture, cells were treated after 24 hours with dimethyl sulfoxide (DMSO, control), docetaxel IC50, GSK461364 IC50, or both in combination. After seven days, or when spheres diameter was > 60 um, formed spheres were counted using a Celigo Imaging Cytometer (Nexcelom, USA). The primary tumorspheres were harvested, dissociated with trypsin and collected. Five hundred dissociated cells were then cultured in ultra-low attachment 96 well plates (Costar®, USA) in serum-free medium for an additional seven days in the absence of drug. Secondary passage tumorspheres were then counted as before.
Giordano A., Liu Y., Armeson K., Park Y., Ridinger M., Erlander M., Reuben J., Britten C., Kappler C., Yeh E, & Ethier S. (2019). Polo-like kinase 1 (Plk1) inhibition synergizes with taxanes in triple negative breast cancer. PLoS ONE, 14(11), e0224420.
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7

Viral Transport Media Formulation

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Viral transport media (VTM) was produced following the US CDC (Atlanta, GA, USA) guidelines, found freely available on their website for formulation of VTM in a laboratory as an alternative to commercial VTM purchases. 500 mL of Hanks Balanced Salt Solution (HBSS) 1X with calcium and magnesium ions (no phenol red) (Fisher Science, Cat. No. SH3058801) was supplemented with 2% heat inactivated fetal bovine serum (Gemini Bioproducts, Cat. No. 100–500). 100 mg of Gentamicin (Gemini Bioproducts, Cat. No. 400-100P) and 500 μg of Amphotericin B (Gemini Bioproducts, Cat. No. 400-104P) was added to the mixture and mixed thoroughly to create a final product of VIRAL TRANSPOT MEDIA, 2% FBS, 100 μg/mL Gentamicin, 0.5 μg/mL Amphotericin B. This viral transport media was used for storage and processing of all swab samples in this manuscript.
Morehouse Z.P., Proctor C.M., Ryan G.L, & Nash R.J. (2020). A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E. Virology Journal, 17, 129.
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8

Preparation of SARS-CoV-2 Virus Stock from Patient Sample

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Before testing, a virus stock of SARS-CoV-2 (USAWA1/2020 strain) sourced from the CDC following its original isolation from an infected patient in Washington state, USA (WA-1 strain BEI #NR-52281 strain), was prepared according to Pelletier and colleagues [12 (link)]. The virus stock was prepared by infecting Vero 76 cells (ATCC CRL-1587) until cytopathic effect (CPE) was visible at two days post-inoculation. The Vero 76 cells were cultured in Minimal Essential Medium (MEM) (Quality Biological), supplemented with 2% (v/v) Fetal Bovine Serum (Sigma), and 50 μg/ml gentamicin (Gemini Bio-products) [12 (link)].
, & Köntös Z. (2021). Efficacy of “Essential Iodine Drops” against Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2). PLoS ONE, 16(7), e0254341.
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9

Maintenance of Planarian Strains

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Hermaphroditic, sexual S. mediterranea were maintained at 18°C in 0.75X Montjuïc salts (Cebrià and Newmark, 2005 (link)) supplemented with 50 μg/mL Gentamicin (Gemini Bio-Products) and fed beef liver paste. Asexual S. mediterranea (clone CIW4; Sánchez Alvarado et al., 2002 (link)) were maintained at 22°C in 1X Montjuïc salts and fed beef liver paste. Planarians were maintained in Ziploc containers and kept in Petri dishes for RNAi experiments. Planarians were starved at least one week before initiating RNAi experiments, in situ hybridizations, or immunostainings.
Khan U.W, & Newmark P.A. (2022). Somatic regulation of female germ cell regeneration and development in planarians. Cell reports, 38(11), 110525.
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10

Lung Isolation and Preparation

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All animal work was performed in accordance with AAALAC guidelines and was approved by the Yale Institutional Animal care and Use Committee (IACUC). Lungs were harvested from adult Sprague Dawley male rats as previously described [25 , 26 (link)]. Briefly, rats were anesthetized with a mixture of 75 mg/kg of ketamine and 5 mg/kg xylazine, and the heart was perfused with Phosphate Buffered Saline (PBS) containing heparin (100 U/mL, Sigma) and sodium nitroprusside (SNP, 10 μg/mL, Sigma) via the right ventricle using gravity-driven flow at a pressure head of 30 cmH2O. After 20 mL had been perfused, the heart, lungs, and trachea were removed en bloc and weighed. Cannulae were then inserted into the trachea, into the pulmonary artery (PA) via the right ventricle, and into the pulmonary vein (PV) via the left atrium.
Lungs were submerged in PBS with 100 U/mL penicillin, 1000 μg/mL streptomycin (both from Gibco), 10 μg/mL amphotericin B, and 200 μg/mL gentamicin (both from Gemini Bioproducts). Lungs were then inflated with 10 mL of air via syringe while submerged to check for pleural leakage due to nicks during explant. An additional 20 mL of PBS/Heparin/SNP was perfused through the PA via syringe while ventilating the lungs. Lungs were lastly either mounted in the bioreactor system as described in Section 2.5 or were decellularized as described in Section 2.6.
Engler A.J., Raredon M.S., Le A.V., Yuan Y., Oczkowicz Y.A., Kan E.L., Baevova P, & Niklason L.E. (2019). Non-Invasive and Real-Time Measurement of Microvascular Barrier in Intact Lungs. Biomaterials, 217, 119313.
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