OCI/AML2 and 3 cells were grown in alpha-MEM (Euroclone, Milan, Italy) supplemented with 20% fetal bovine serum, 100 U/mL penicillin and 10 µg/mL streptomycin sulfate. Jurkat and HL-60 cells were grown in RPMI-1640 medium (Euroclone, Milan, Italy); all cells were supplemented with 10% fetal bovine serum (FBS, EuroClone, Milan, Italy), L-glutamine (2 mM) (Euroclone, Milan, Italy), penicillin (100 U/mL) and streptomycin (100 mg/mL) (Euroclone, Milan, Italy), and were cultured in a humidified incubator, at 37 °C, in a 5% v/v CO2 atmosphere. All chemical reagents used for treatments were supplied from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
Streptomycin sulfate
Manufactured by Euroclone
Sourced in Italy
Streptomycin sulfate is a white, crystalline powder that is soluble in water and has a bitter taste. It is a broad-spectrum antibiotic derived from the bacterium Streptomyces griseus.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Euroclone (5 mentions)
Fetal bovine serum (fbs), by Euroclone (4 mentions)
Glutamine, by Euroclone (3 mentions)
Trypsin, by Harvard Bioscience (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Harvard Bioscience (2 mentions)
Streptomycin, by Euroclone (1 mentions)
Alpha mem, by Euroclone (1 mentions)
Rpmi 1640 medium, by Euroclone (1 mentions)
L glutamine, by Euroclone (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Penicillin g, by Euroclone (1 mentions)
Amphotericin b, by Euroclone (1 mentions)
Penicillin g sodium salt, by Euroclone (1 mentions)
Sodium pyruvate, by Euroclone (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
7 protocols using streptomycin sulfate
1
Cell Culture Conditions for Hematological Malignancies
2
Optimizing 4D Bioreactor for Neurodegeneration Research
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SH-SY5Y human neuroblastoma cells (ATCC® code CRL-2266TM) are a widely used model for neurodegenerative disorders. They were cultured at 37°C and 5% CO2 in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate (EuroClone, Pero, Italy) and split twice a week.
To adapt our 4D microfluidic bioreactor for use in the assessment of potential therapeutic strategies against neurodegeneration, we have focused on the optimization of the following experimental protocols (Figure2A ):
Finally, we have focused on the demonstration that SH-SY5Y cells retain their basic capacity to secrete PRGN in exosomes once recovered from the 4D bioreactor (Figure2B ).
To adapt our 4D microfluidic bioreactor for use in the assessment of potential therapeutic strategies against neurodegeneration, we have focused on the optimization of the following experimental protocols (Figure
Finally, we have focused on the demonstration that SH-SY5Y cells retain their basic capacity to secrete PRGN in exosomes once recovered from the 4D bioreactor (Figure
3
Culturing MCF10 Breast Cell Lines
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MCF10 human breast cell lines, MCF10A and MCF10CA, were cultured in DMEM/F12 medium (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 5% horse serum (Invitrogen, Carlsbad, CA, USA), 2 mM glutamine (EuroClone, Pavia, Italy), 100 µg/mL streptomycin sulfate, 100 U/mL penicillin (EuroClone), 0.5 µg/mL epidermal growth factor (EGF, Sigma), 0.5 µg/mL hydrocortisone (Sigma), 0.1 µg/mL cholera toxin (Sigma), and 10 µg/mL insulin (Sigma). Both cell lines were passaged twice a week at the following split ratios (MCF10CA 1:20; MCF10A 1:25) using 0.05% trypsin (w/v) 0.02% EDTA (w/v) (Biochrom AG, Berlin,Germany). For experiments with the EV isolation, serum was purified by centrifugation at 110,000× g for 16 h and sterile-filtered with 0.22 µm syringe filters (Guangzhou Jet Bio-Filtration Co., Ltd., Guangdong, China).
4
Isolation and Culture of Rat Bone Marrow MSCs
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MSCs were obtained from the bone marrow of Sprague-Dawley rats (Envigo) by flushing the femur and tibia diaphysis with 2 mL/bone of alpha MEM with 2 mM Glutamine and antibiotics (100 U/mL Penicillin G and 100 µg/mL Streptomycin Sulfate) (Euroclone). MSCs were cultured in a humidified incubator at 37 °C with 5% CO2 in alpha-MEM medium plus 2 mM Glutamine, antibiotics and 20% fetal bovine serum (Euroclone).
5
MCF-7 Breast Cancer Cell Culture
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The human breast adenocarcinoma cell line, MCF-7, was cultured in minimum essential medium alpha (MEMα, EuroClone, Pavia, Italy) supplemented with 5% fetal bovine serum (FBS, HyClone, Thermo Scientific, Epsom, UK), 2 mM glutamine (EuroClone), 50 μg/mL streptomycin sulfate, and 50 U/mL penicillin (EuroClone). Cells were passaged twice a week at a 1 : 5 split ratio using 0.05% trypsin (w/v) 0.02% EDTA (w/v) (Biochrom AG, Berlin, Germany).
6
Evaluating Vaccinal Infectious Virus
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To evaluate the potential presence of vaccinal infectious virus, virus isolation in cell cultures was performed for all vaccinal strains from tissue samples, as previously described (Ogbu et al. 2021 (link)). Briefly, samples were inoculated on subconfluent A-72 cell monolayers and left in contact for 30 min at 37 °C and 5% CO2. Culture medium, Eagle’s Minimum Essential Medium (EMEM) (Sigma–Aldrich®, Milan, Italy) supplemented with an antibiotic and antimycotic solution (100 U/mL penicillin G sodium salt, 0.1 mg/mL streptomycin sulfate, 0.25 µg/mL amphotericin B; EuroClone®, Milan, Italy), 1% sodium pyruvate (EuroClone®, Milan, Italy) and 10% fetal bovine serum (FBS; EuroClone®, Milan, Italy), was then added and the incubation was performed at 37 °C and 5% CO2 for 6–7 days. Inoculated cells were monitored daily and, according to standard laboratory procedures, four additional blind passages were carried out before considering virus isolation as unsuccessful. Viral growth was evaluated by the detection of cytopathic effect (CPE). Moreover, inoculated cell monolayers were subjected to three cycles of freeze-thawing, centrifuged at 1,500 x g for 15 min at 4 °C, and the collected supernatants were tested for CPV-2 DNA by PCR assays (Touihri et al. 2009 (link)) to confirm the presence of infectious virus.
7
Maintenance of K562 Erythroleukemic Cells
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K562 cell line, a human erythroleukemic cell line which does not express MHC class I molecules, was provided by Dr. Alessandro Zerbini from the Azienda Ospedaliero-Universitaria of Parma, Italy and maintained in RPMI 1640 (Gibco, ThermoFisher) supplemented with 10% FBS, 100 U/mL penicillin (Euroclone), and 100 μg/mL streptomycin sulfate (Euroclone) at 37°C, 5% CO2.
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