The Benin 97/1 genotype I (genome reference AM712239 ) virulent field isolate has been previously described (60 (link)). The Benin 97/1 isolate was cultured in PBMs in Earle's balanced salt solution (EBSS) (Sigma), supplemented with 4 mM Hepes, 10% heat-inactivated porcine serum (BioSera, France), 100 IU/mL penicillin, and 100 µg/mL streptomycin (61 (link)). Titration of the viruses was carried out by hemadsorption assay (presented as HAD50/mL) or by immunofluorescence using antibodies against ASFV early protein p30/CP204L (presented as TCID50/mL), calculated using the Spearman and Karber formula.
Hepes
Manufactured by Biosera
Sourced in France
HEPES is a buffering agent commonly used in cell culture and biochemical applications. It helps maintain a stable pH environment for sensitive biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12 medium, by Biosera (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Heparin, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
T 25 ultra low attachment flask, by Corning (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Biosera (1 mentions)
Streptomycin, by Biosera (1 mentions)
Leukemia inhibitory factor, by ProSpec (1 mentions)
Amphotericin b, by Biosera (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi glutamax, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Hepes, by Eurobio Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Merck Group (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
5 protocols using hepes
1
ASFV Benin 97/1 Genotype I Propagation
2
Gastric Cancer Tumor Sphere Culture
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Fresh tumor specimens (0.3-4 mL) obtained from GC patients (Table 1 ) were disaggregated into single cell suspension in DMEM/F12 medium (Biosera, France) containing collagenase type I (Gibco, USD)
(300 U.mL-1), penicillin (Biosera) (500 U.mL-1), streptomycin (Biosera) (500 mg.mL-1), and amphotericin B (Biosera) (1.25 mg.mL-1) at 37 °C for 2 hr. The cells (1 × 105 cell.mL-1) were cultivated in epidermal growth factor (EGF) 20 ng.mL-1 (Gibco), basic fibroblast growth factor (bFGF) 10 ng.mL-1 (Gibco), leukemia inhibitory factor (LIF) 10 ng.mL-1 (ProSpec, Israel), heparin 4 μg.mL-1 (Sigma-Aldrich, Germany), B-27 supplement 2% (Gibco), penicillin 100 U.mL-1, and streptomycin 100 μg.mL-1, and HEPES 8 mM (Biosera) in DMEM/F12 medium in T-25 ultra-low attachment flask (Corning, USA) at 37 °C for 1-2 months in a humidified 5% CO2 incubator to generate spheres.
(300 U.mL-1), penicillin (Biosera) (500 U.mL-1), streptomycin (Biosera) (500 mg.mL-1), and amphotericin B (Biosera) (1.25 mg.mL-1) at 37 °C for 2 hr. The cells (1 × 105 cell.mL-1) were cultivated in epidermal growth factor (EGF) 20 ng.mL-1 (Gibco), basic fibroblast growth factor (bFGF) 10 ng.mL-1 (Gibco), leukemia inhibitory factor (LIF) 10 ng.mL-1 (ProSpec, Israel), heparin 4 μg.mL-1 (Sigma-Aldrich, Germany), B-27 supplement 2% (Gibco), penicillin 100 U.mL-1, and streptomycin 100 μg.mL-1, and HEPES 8 mM (Biosera) in DMEM/F12 medium in T-25 ultra-low attachment flask (Corning, USA) at 37 °C for 1-2 months in a humidified 5% CO2 incubator to generate spheres.
3
Cytotoxic Effects of Compounds on Cancer Cells
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Two different human breast cancer cell lines (MCF-7 and MDA-MB-231), lung cancer cell line (A-549) and normal cell (Human Dermal Fibroblast) were purchased from Pasture Institute of Iran, Tehran, Iran. The medium of RPMI 1640 (PAA, Germany) including sodium bicarbonate and N-hydroxyethylpiperazone-n-2-ethanesulfonic Acid (HEPES, Biosera, England) was used to maintain the cell lines. The medium was enriched with fetal bovine serum (FBS; Gibco, USA) and antibiotics. Then, incubated in air atmosphere enriched 5% CO2 at 37 °C. The cytotoxic activity of all fractions and compounds were examined by the MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, USA) assay.
4
Cell Culture Conditions for Murine Cell Lines
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MutuDC 1940 (obtained from Hans Acha Orbea) cells were grown in IMDM (Sigma-Aldrich, #I3390-500ML), supplemented with 8% FCS (Biosera or Eurobio), 10 mM HEPES (#15630080), 2 mM glutamax (#35050061), 100 U/mL penicillin, 100 μg/mL streptomycin (#15140122) and 50 μM β-mercaptoethanol (#31350010) (all from Life Technologies). B3Z hybridomas were grown in RPMI-glutamax (Gibco, #61870010), supplemented with 10% FCS (Biosera or Eurobio), 10 mM HEPES, 1 mM sodium pyruvate (#11360070), 1% MEM non-essential amino acids (#11140035), 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM β-mercaptoethanol (all from Life Technologies). HEK293T were grown in DMEM-glutamax (Gibco, #31966021), supplemented with 10% FCS (Biosera or Eurobio).
All cell lines used tested negative from Mycoplasma by PCR.
Splenic dendritic cells were grown in RPMI-glutamax (Gibco, #61870010), supplemented with 10% FCS (Biosera), 10 mM HEPES, 1 mM sodium pyruvate (#11360070), 1% MEM non-essential amino acids (#11140035), 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM β-mercaptoethanol (all from Life Technologies).
All cell lines used tested negative from Mycoplasma by PCR.
Splenic dendritic cells were grown in RPMI-glutamax (Gibco, #61870010), supplemented with 10% FCS (Biosera), 10 mM HEPES, 1 mM sodium pyruvate (#11360070), 1% MEM non-essential amino acids (#11140035), 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM β-mercaptoethanol (all from Life Technologies).
5
Isolation and Characterization of Chemotherapy-Enriched Cancer Stem Cells
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Chemotherapy-enriched sphere-forming cells with cancer stem cell properties isolated from a 54-year-old male patient with GC (T3N0M0) were previously identi ed as described [22, (link)23] (link). The ethics committee of MUMS approved the investigation and written informed consent was acquired from patient (930607).
The cells were cultured in DMEM/F12 medium (Biosera, France) containing eight mM HEPES (Biosera), 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco, Carlsbad, CA) at 37°C in a humidi ed atmosphere containing 5% CO2 for ve days, with medium changed daily. Then the cells were subcultured every ve days for 250 days.
The cells were cultured in DMEM/F12 medium (Biosera, France) containing eight mM HEPES (Biosera), 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco, Carlsbad, CA) at 37°C in a humidi ed atmosphere containing 5% CO2 for ve days, with medium changed daily. Then the cells were subcultured every ve days for 250 days.
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