L. (V.) braziliensis (MCAN/BR/98/R619) was routinely isolated from hamsters’ skin lesions. The animals were infected on the dorsal hind paw with 5 × 106 promastigotes of L. (V.) braziliensis at the stationary phase. The infection was maintained for 30–40 days, and the skin of the lesion was surgically removed and homogenized with 1 ml of Phosphate-buffered saline (PBS) using a tissue grinder. The cell suspension was incubated with Schneider’s medium (Sigma-Aldrich) containing 20% inactivated fetal bovine serum (FBS) at 27°C. Promastigotes were maintained with weekly passages in Schneider’s medium with 20% FBS and 100 µg/ml gentamicin (Schering-Plough, Kenilworth, New Jersey, USA) at 27°C. Parasites were used for up to five passages in culture, at which time they were reisolated from infected hamsters.
Gentamicin
Manufactured by Merck & Co
Sourced in Brazil, United States, Japan
Gentamicin is a broad-spectrum antibiotic used in laboratory settings. It is a type of aminoglycoside antibiotic that works by inhibiting protein synthesis in bacterial cells, leading to their death. Gentamicin is commonly used in cell culture media to prevent bacterial contamination.
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Lab products found in correlation
Penicillin, by Merck & Co (6 mentions)
Penicillin, by Merck Group (5 mentions)
Streptomycin, by Merck Group (5 mentions)
Schneider s insect medium, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Cultilab (4 mentions)
75 cm2 cell culture flask, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle s medium high glucose, by Merck Group (3 mentions)
Amphotericin b, by Merck Group (3 mentions)
Fungizone, by Merck & Co (2 mentions)
Amphotericyn b, by Merck Group (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
Fungizone amphotericin b, by Cristália (2 mentions)
Cytometric bead assay kit, by BD (2 mentions)
Dimethyl sulfoxide (dmso), by Avantor (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Nystatin, by Merck Group (2 mentions)
Bovine serum albumin fraction 5, by Roche (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
32 protocols using gentamicin
1
Isolation and Maintenance of L. braziliensis
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L. (V.) braziliensis (MCAN/BR/98/R619) was routinely isolated from hamsters’ skin lesions. The animals were infected on the dorsal hind paw with 5 × 106 promastigotes of L. (V.) braziliensis at the stationary phase. The infection was maintained for 30–40 days, and the skin of the lesion was surgically removed and homogenized with 1 ml of Phosphate-buffered saline (PBS) using a tissue grinder. The cell suspension was incubated with Schneider’s medium (Sigma-Aldrich) containing 20% inactivated fetal bovine serum (FBS) at 27°C. Promastigotes were maintained with weekly passages in Schneider’s medium with 20% FBS and 100 µg/ml gentamicin (Schering-Plough, Kenilworth, New Jersey, USA) at 27°C. Parasites were used for up to five passages in culture, at which time they were reisolated from infected hamsters.
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L. (V.) braziliensis (MCAN/BR/98/R619) was routinely isolated from hamsters’ skin lesions. The animals were infected on the dorsal hind paw with 5 × 106 promastigotes of L. (V.) braziliensis at the stationary phase. The infection was maintained for 30–40 days, and the skin of the lesion was surgically removed and homogenized with 1 ml of Phosphate-buffered saline (PBS) using a tissue grinder. The cell suspension was incubated with Schneider’s medium (Sigma-Aldrich) containing 20% inactivated fetal bovine serum (FBS) at 27°C. Promastigotes were maintained with weekly passages in Schneider’s medium with 20% FBS and 100 µg/ml gentamicin (Schering-Plough, Kenilworth, New Jersey, USA) at 27°C. Parasites were used for up to five passages in culture, at which time they were reisolated from infected hamsters.
2
Purification and Culture of Mimiviruses
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Acanthamoeba polyphaga mimivirus, a prototype of the Mimiviridae family, and APMV M4, a strain derived from APMV after 150 passages in amoeba culture (Boyer et al., 2011 (link)), were kindly provided by Dr. Didier Raoult (Aix Marseille Université, France). The Brazilian mimivirus isolates Kroon virus, Oyster virus, and Samba virus were produced and purified as previously described (La Scola et al., 2003 (link)). Briefly, A. castellanii (ATCC 30010) cells were grown in 75-cm2 cell culture flasks (Nunc, USA) in peptone-yeast extract-glucose (PYG) medium (Visvesvara and Balamuth, 1975 (link)) supplemented with 7% fetal calf serum (FCS, Cultilab, Brazil), 25 mg/mL fungizone (amphotericin B, Cristalia, Brazil), 500 U/mL penicillin, and 50 mg/mL gentamicin (Schering-Plough, Brazil). After reaching confluence, the amoebas were infected and incubated at 32°C until cytopathic effects were observed. Supernatants from the infected amoebas were collected and filtered through a 0.8-μm filter to remove cell debris. The viruses were purified by centrifugation through a sucrose cushion (24%), suspended in phosphate-buffered saline (PBS), and stored at -80°C. For the gene expression experiments, A. castellanii was maintained in PYG medium in the absence or in the presence of 7% FCS at 32°C or in Page’s amoeba saline (PAS) at 32°C to induce starvation.
3
Optimized Stem Cell Culture Media
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Basal medium consisted of Dulbecco’s modified Eagle’s medium-high glucose (Sigma-Aldrich, USA) supplemented with 5 mM sodium bicarbonate (Cinética Química Ltda, Brazil), penicillin (100 units/mL; Sigma-Aldrich), streptomycin (0.1 mg/mL; Sigma-Aldrich), amphotericyn B (0.25 μg/mL; Sigma-Aldrich), gentamicin (60 mg/L; Schering-Plough, USA), and 10% of either aHS or FBS (Cripion Biotecnologia Ltda, Brazil). Adipogenic medium consisted of basal medium with 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), 200 μM indomethacin (Sigma-Aldrich), 1 μM dexamethasone (Aché, Brazil), and 10 μM insulin (Eli Lilly and Company, USA). Osteogenic medium consisted of basal medium with 50 μg/mL ascorbate-2-phosphate (Ecibra, Brazil), 10 mM β-glycerophosphate (Sigma-Aldrich), and 0.1 μM dexamethasone (Aché). Chondrogenic medium consisted of basal medium with 1 mM dexamethasone (Aché), 125 μg/mL bovine serum albumin (PAA, Austria), 1 mM pyruvate (Sigma-Aldrich), 200 U/mL insulin (Eli Lilly and Company), 3.25 μg/mL transferrin (Wako, Brazil), 0.01 μg/mL transforming growth factor-β1 (Sigma-Aldrich), 5 mg/mL ascorbate-2-phosphate (Ecibra) and a reduced concentration of serum supplements - 1% aHS or 1% FBS [5 (link)].
4
Stereotactic Lesions of the Accumbens Core
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Stereotactic lesions of the AcbC were made using a standard stereotaxic apparatus (David Kopf Instruments). Rats were first injected with pentobarbital sodium (i.p.; 50 mg/kg body weight, Dainippon Sumitomo Pharma Co., Ltd) dissolved in saline and then maintained on anesthesia with isoflurane (Foren; Abbott Japan Co., Ltd.) for the surgical procedure. Bilateral lesions were made by infusing ibotenic acid (0.6 µl/hemisphere of 5 mg/ml ibotenic acid in PBS; Sigma Aldrich, Japan) through a stainless steel needle (200 µm outer diameter) attached to the stereotaxic device. Coordinates for injection sites were as follows: AP, +1.9 mm rostral to bregma; ML, ±1.7 mm lateral to the midline; V, −6.3 mm ventral to the dura mater. Sham-lesioned rats received injections of the same volume (0.6 µl/hemisphere) of PBS at these coordinates. The needle was left in place for 15–20 min to allow the toxin to diffuse sufficiently within the target sites. Rats were allowed to recover for 7 days following the lesion surgery. For the first 4 days after surgery, rats were given daily i.p. injections of antibiotic solution (0.4 ml/day of 1 mg/ml gentamicin in saline; Schering-Plough).
5
Purification and Isolation of APMV Particles
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APMV particles were isolated and purified from infected amoebae as previously described [16] (link). Briefly, Acanthamoeba castellanii (ATCC 30234) were grown in 75 cm2 cell culture flasks (Nunc, USA) in PYG (peptone-yeast extract-glucose) medium supplemented with 7% fetal calf serum (FCS, Cultilab, Brazil), 25 mg/ml Fungizone (amphotericin B, Cristalia, São Paulo, Brazil), 500 U/ml penicillin and 50 mg/ml gentamicin (Schering-Plough, Brazil). After reaching confluence, the amoebas were infected with APMV and incubated at 37°C until the appearance of cytopathic effects. APMV-rich supernatants from the infected amoeba were collected and filtered through a 0.8-micron (Millipore, USA) filter to remove amoeba debris. The viruses were then purified using a Gastrografin gradient (45–36–28%) [5] (link), suspended in PBS and stored at −80°C.
6
Isolation and Purification of Tupanvirus
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Tupanvirus soda lake (TPVsl) was isolated from a soda lake sample from the Pantanal region in Brazil and was produced and purified as previously described (Abrahão et al., 2018 (link)). Briefly, A. castellanii (ATCC 30010) cells were grown in 75 cm2 cell culture flasks (Nunc, United States) in peptone–yeast extract–glucose (PYG) medium (Visvesvara and Balamuth, 1975 (link)) supplemented with 25 mg/mL fungizone (Amphotericin B, Cristalia, Brazil), 500 U/mL penicillin, and 50 mg/mL gentamicin (Schering-Plough, Brazil). After reaching confluence, the amoebae were infected at a multiplicity of infection (m.o.i) of 0.1 and incubated at 32°C until cytopathic effects (CPE) were observed. Supernatants from the infected amoebae were collected and filtered through a 0.8 μm filter to remove cell debris. The viruses were purified by centrifugation through a sucrose cushion (22%), suspended in phosphate-buffered saline (PBS), and stored at -80°C.
7
Cultivation and Characterization of L. infantum Promastigotes
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L. infantum (MCAN/BR/2008/OP46) promastigotes, which were isolated from an infected dog at Governador Valadares, MG, Brazil, and characterized by molecular techniques and hamster infection23 (link),24 (link). They were maintained at 26 °C in NNN-LIT medium (Sigma) supplemented with 20% heat-inactivated fetal calf serum (Criprion Biotech) and 40 μg gentamicin ml−1 (Schering-Plough). For all assays, promastigotes in the stationary phase of growth (5–6 days) were washed twice in PBS (pH 7.2) at 1900 x g for 13 min at room temperature and counted using a hemocytometer.
8
Isolation and Culture of hASCs
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Human adipose tissue was harvested from healthy patients who had abdominal reduction surgery. All the donors provided written informed consent. The approval number in the Ethics Committee in Research from Federal University of Minas Gerais is—ETIC 0023.0.203.000-11. The hASCs were obtained and cultured as described previously by Zuk et al. [18 (link)]. The cells were cultured in basal medium Dulbecco's modified Eagle's medium-high glucose (Sigma-Aldrich) supplemented with 5 mM sodium bicarbonate, penicillin (100 units/mL), streptomycin (0.1 mg/mL), amphotericin B (0.25 μg/mL) (Sigma-Aldrich), gentamicin (60 mg/L, Schering-Plough), and 10% of fetal bovine serum (FBS) (Cripion Biotecnologia LTDA, Brazil) at 37°C in a 5% CO2 humidified atmosphere. And the hASCs were cultured under basal medium with SWCNT-MA suspension (diluted 1/5 or diluted 1/10 in basal medium) to perform the assays.
9
Viral Isolation and Characterization Protocol
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African green monkey kidney BSC-40 and VERO cells (American type cell culture—ATCC) were maintained in 5% CO2 atmosphere at 37 °C in Eagle’s Minimum Essential Medium (MEM) (Gibco BRL, Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS) (Cultilab, Brazil), 25 µg/mL fungizone (Amphotericin B) (Cristália, São Paulo, Brazil), 500 U/mL penicillin (Cristália, São Paulo, São Paulo, Brazil) and 50 µg/mL gentamicin (Schering-Plough, São Paulo, Brazil). VERO cells were used for viral isolation from clinical specimens and growth. The BSC-40 cells were used for viral clones purification, plaque phenotype, comet phenotype and growth curve assays. Only typical poxviruses cytopathic effects were visualized after inoculation of clinical samples in both cell systems. The VACV Western Reserve (VACV-WR) was gently provided by Dr C. Jungwirth (Universität Würzburg, Würzburg, Germany) and was used as a virulent control in mice assays.
10
Characterization of Cancer Cell Lines
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The following cell lines were used: human melanoma cell lines (SKMel 28 and A2058), originally provided by Dr. Alan N. Houghton of Memorial Sloan Kettering Cancer Center, NY; murine melanoma (B16F10-Nex2), a subclone of the B16F10 cell line obtained at the Experimental Oncology Unit (UNONEX), Federal University of São Paulo; human cervical cancer (HeLa), human umbilical vein endothelial cells (HUVEC), mouse fibroblasts (3T3) and human foreskin fibroblast (HF) cell lines were provided by Ludwig Institute for Cancer Research, São Paulo and Dr. Luiz F. Lima Reis, Hospital Sirio-Libanez, São Paulo, Brazil The U87-MG glioblastoma cell line was provided by Dr. Osvaldo K. Okamoto, University of São Paulo. Murine, syngeneic, colorectal adenocarcinoma cell (CT26) and murine pancreatic cells (PANC) were provided by Dr. Guillermo Mazzolini from the School of Medicine of Austral University, Derqui-Pilar, Buenos Aires, Argentina. Tumor cells were cultured at 37°C in a humidified atmosphere containing 5% CO2, in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10mM N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid (Hepes) (Sigma, St. Louis, MO), 24mM sodium bicarbonate (Sigma), 40mg/l gentamicin (Schering-Plough, São Paulo, Brazil), pH 7.2 and 10% fetal calf serum (Invitrogen). Human HF, mouse CT26 and 3T3 cells were maintained in DMEM supplemented as for the RPMI-1640 medium.
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