Experiments were approved by the ethics committee (CEUA, Instituto Evandro Chagas, 42/2019). Feather pulp samples were collected from a male of M. americana maintained at Parque Mangal das Garças (Belém, PA, Brazil) and transported in ice to the laboratory at Instituto Evandro Chagas. Cell culture was performed according to Sasaki et al. [26 (link)] with modifications. The material was extracted from the feather in a clean Petri dish and mechanically dissociated with the use of scalpel blades before incubation in collagenase solution (0.0465 g/mL DMEN) for approximately 1 h. Afterward, the material was centrifuged, and the supernatant was discharged and substituted by 5 mL of DMEM (Sigma-Aldrich, MO, USA) supplemented with 10 % bovine fetal serum (Gibco, Waltham, MA, USA) and 5% Aminiomax (Invitrogen, Carlsbad, CA, USA). Culture flasks were maintained at 37 °C in a 5% CO2 incubator. Chromosomes were obtained after exposition to colcemid (0.0016 %, 1 h), hypotonic treatment (0.075 M KCl, 40 min), and fixation and washes with Carnoy fixative (3 methanol:1acetic acid), following standard procedures. Chromosome suspensions were kept in a freezer at −20 °C.
Bovine fetal serum
Manufactured by Thermo Fisher Scientific
Sourced in United States, Brazil, Germany
Bovine fetal serum is a cell culture supplement derived from the blood of bovine fetuses. It provides essential nutrients, growth factors, and other components to support the growth and maintenance of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
High glucose dmem, by Thermo Fisher Scientific (3 mentions)
Streptomycin sulfate, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
96 well plate, by Corning (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Amphotericin b, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Penicillin streptomycin neomycin antibiotic mixture, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Penicillin g, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
47 protocols using bovine fetal serum
1
Cytogenetic Analysis of M. americana
2
Neonatal Rat Cardiomyocyte Primary Culture
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Primary cultures of cardiomyocytes were prepared from neonatal rat hearts (1–2 days-old) according to the method previously described [30 (link)]. This study has been specifically approved by the ethics committee Institutional Care and Use Committee (IACUC) of the School of Medicine, National Autonomous University of Mexico. Cells were grown in DMEM (Dulbecco´s modified Eagle´s medium from Invitrogen, CA, USA) supplemented with 10% bovine fetal serum (Gibco, MA, USA), kanamycin (60 mg/mL) (Sigma-Aldrich, MO, USA), penicillin (10 U/mL) (Gibco, MA, USA), streptomycin (10 mg/mL) (Gibco, MA, USA), amphotericin B (0.025 mg/mL) (Gibco, MA, USA) and nystatin (10 U/mL) (Sigma-Aldrich, MO, USA).
3
Peptide Synthesis and Cell Assays
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Rink Amide Resin, Fmoc-Arg(Pbf)-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-6-Ahx-OH, Fmoc-Lys(Fmoc)-OH, Triton-X,
piperidine, N,N-dimethylformamide
(DMF), dichloromethane (DCM), dicyclohexylcarbodiimide (DCC), 1-hydroxy-6-chloro-benzotriazole
(6-Cl-HOBt), ninhydrin, potassium cyanide (KCN), ethanol, pyridine,
phenol, trifluoroacetic acid (TFA), ethyl ether, triisopropylsilane
(TIS), ethanedithiol (EDT), acetonitrile (ACN), methanol (MeOH), and
phase extraction columns solid Supelco , Trypsin–EDTA solution,
and pepsin 2500 units mg–1 were purchased from Sigma-Aldrich
(St. Louis, MO, USA). The primary cell culture of fibroblasts was
obtained from the foreskin by the Laboratory of Cellular Physiology
of the National University of Colombia, and the fibroblasts were cultured
and frozen. Annexin V, Alexa Fluor 488 conjugate was purchased from
Invitrogen (Eugene, Oregon). The RPMI 1640 culture medium and trypsin
were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bovine fetal
serum, penicillin, and streptomycin were purchased from Gibco (Waltham,
MA, USA). A calcium assay kit and a Fluorescein CaspGLOW Active Caspase-8
or Caspase-9 Staining Kit were obtained for BD Biosciences (Tor- reyana
Rd., San Diego, CA 92121, USA) and Invitrogen Thermo Fisher Scientific
(Waltham, Massachusetts, USA), respectively.
Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-6-Ahx-OH, Fmoc-Lys(Fmoc)-OH, Triton-X,
piperidine, N,N-dimethylformamide
(DMF), dichloromethane (DCM), dicyclohexylcarbodiimide (DCC), 1-hydroxy-6-chloro-benzotriazole
(6-Cl-HOBt), ninhydrin, potassium cyanide (KCN), ethanol, pyridine,
phenol, trifluoroacetic acid (TFA), ethyl ether, triisopropylsilane
(TIS), ethanedithiol (EDT), acetonitrile (ACN), methanol (MeOH), and
phase extraction columns solid Supelco , Trypsin–EDTA solution,
and pepsin 2500 units mg–1 were purchased from Sigma-Aldrich
(St. Louis, MO, USA). The primary cell culture of fibroblasts was
obtained from the foreskin by the Laboratory of Cellular Physiology
of the National University of Colombia, and the fibroblasts were cultured
and frozen. Annexin V, Alexa Fluor 488 conjugate was purchased from
Invitrogen (Eugene, Oregon). The RPMI 1640 culture medium and trypsin
were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bovine fetal
serum, penicillin, and streptomycin were purchased from Gibco (Waltham,
MA, USA). A calcium assay kit and a Fluorescein CaspGLOW Active Caspase-8
or Caspase-9 Staining Kit were obtained for BD Biosciences (Tor- reyana
Rd., San Diego, CA 92121, USA) and Invitrogen Thermo Fisher Scientific
(Waltham, Massachusetts, USA), respectively.
4
PBMC Isolation from Heparinized Blood
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Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood by density gradient centrifugation using Ficoll-Hypaque (GE Healthcare). Cells were washed twice in saline and were resuspended at the desired concentration in RPMI 1640 supplemented with 10% bovine fetal serum (both produced by Gibco, Grand Island, NY, USA) plus antibiotics (complete RPMI).
5
Synthesis and Characterization of H2bdtc
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BZN, a product manufactured by Lafepe, Brazil, was used as a reference drug. The synthesis of H2bdtc has been described previously [21] (link). RPMI Medium 1640 supplemented with 5% bovine fetal serum (GIBCO, Grand Island, NY, USA), 100 IU mL−1 penicillin G, and 100 mg mL−1 streptomycin (Gibco-BRL, Grand Island, NY, USA) was employed. Dimethyl sulfoxide (DMSO) and propidium iodide (PI) were obtained from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Stearic acid and sodium taurodeoxycholate were purchased from Sigma-Aldrich (St. Louis, MO, USA), Lipoid S 100 (soya lecithin) was acquired from Lipoid (Ludwigshafen, KOLN, Germany), and Amicon Ultra 15, MWCO 100 K, was provided by Millipore (Billerica, MA, USA).
6
Culture of Human Cell Lines
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Human CRC cells lines (HCT116 and CACO-2) and Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from the Chinese Academy of Sciences (Shanghai) and cultured in RPMI 1640 medium (Gibco, United States) containing 10% bovine fetal serum (Gibco, United States) in an incubator set to 37°C and 5% CO2.
7
Cultivation and Virulence of N. fowleri
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N. fowleri ATCC 30,808 trophozoites were cultured in axenic conditions at 37 °C in bactocasitone broth (Difco, Le Pont de Claix, France), supplemented with 10% bovine fetal serum (Gibco, Grand Island, NY, USA). Trophozoites were chilled and harvested during the logarithmic phase (72 h). After centrifuging at 1500× g for 10 min, amoebas were washed 3 times with phosphate-buffered saline (PBS) and counted in a hemocytometer. The virulence of N. fowleri was reactivated by serial passage through mice, as described previously [5 (link)]. Only freshly recovered virulent amoebae were used for all experiments.
8
Cell Viability Assay Protocol
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Tripan blue, sodium bicarbonate, Chl-A of spinach, DMEM, P407 (Pluronic® F-127), low-molecular-weight CS were purchased from Sigma Aldrich (St. Louis, MO, USA). Penicillin and streptomycin, bovine fetal serum, and tripsine-EDTA were acquired from Gibco (Waltham, MA, USA). MTT was purchased from Invitrogen (Waltham, MA, USA). Other materials and reagents used in this study were analytical grade and/or suitable for use on cells culture.
9
Cytological and Culturing Leishmania
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Cytological examinations were performed in smears of freshly obtained fine needle aspirates of bone marrow and/or lymph nodes. Smears were stained with a modified Romanowsky staining rapid test kit (Panótico Rápido, Laborclin, Brazil) and analyzed under optical microscopy. A positive cytological result was given by the finding of amastigote forms within cells.
For isolation of Leishmania sp. in culture medium, freshly obtained spleen aspirates were inoculated into biphasic Novy-MacNeal-Nicolle (NNN) medium with Schneider’s liquid phase (Sigma-Aldrich Inc., USA) supplemented with 20% bovine fetal serum (Gibco, USA) and 50 μg/mL gentamicin, as standardized previously [23 (link), 26 (link)]. The cultures were kept in a BOD chamber at 23°C and examined weekly under optical microscopy for 30 days. Positive culture results were identified by the visualization of moving promastigote forms.
For isolation of Leishmania sp. in culture medium, freshly obtained spleen aspirates were inoculated into biphasic Novy-MacNeal-Nicolle (NNN) medium with Schneider’s liquid phase (Sigma-Aldrich Inc., USA) supplemented with 20% bovine fetal serum (Gibco, USA) and 50 μg/mL gentamicin, as standardized previously [23 (link), 26 (link)]. The cultures were kept in a BOD chamber at 23°C and examined weekly under optical microscopy for 30 days. Positive culture results were identified by the visualization of moving promastigote forms.
10
Human Hepatocellular Carcinoma Cell Culture
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Human hepatocellular carcinoma cell lines HepG2 and Huh7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, United States) containing 10% bovine fetal serum (Gibco) and were maintained at 37°C with 5% CO2 supply. Transient gene knockdown was achieved by transfecting cells with specific small-interfering RNAs using the Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen, United States).
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