After 0 and 1440mins of 169MHz RF exposure, 1.5×104/cm2 cells were seeded and osteogenic (Dulbecco Modified Eagle Medium (DMEM) (Microgem), 10% FBS (Microgem), 0.05 mM ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone) or adipogenic (DMEM) (Microgem), 10% Horse Serum (Microgem), 1 mM dexamethasone, 10 µg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 200 µM indomethacin) or chondrogenic (DMEM) (Microgem), 10% FBS (Microgem), 100 IU/mL penicillin (Microgem), 100 mg/mL streptomycin (Microgem), 50 nM ascorbate-2-phosphate, 0.1 mM dexamethasone, and 10 ng/mL human transforming growth factor (hTGF)-β1 (PeproTech, Inc., Rocky Hill, NJ, USA) differentiation was performed. Not differentiated cells were cultivated in DMEM medium (Microgem) supplemented with 10% FBS (Microgem). All conditions were performed for 21 days, renewing the medium every 3 days. Osteogenic differentiation was evaluated by alizarin red S staining, adipogenic differentiation by Oil Red O staining and chondrogenic differentiation by Alcian blue staining as previously described.21 (link) Where not specified otherwise, all reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Fetal bovine serum (fbs)
Manufactured by Microgem
Sourced in Italy, United States
FBS is a type of cell culture supplement used in laboratory research. It is derived from the blood of fetal bovine (calf) and provides a range of nutrients, growth factors, and other components to support the growth and proliferation of cells in vitro. FBS is a widely used component in cell culture media formulations.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Microgem (4 mentions)
Penicillin, by Microgem (3 mentions)
Streptomycin, by Microgem (3 mentions)
L glutamine, by Microgem (3 mentions)
Penicillin streptomycin, by Microgem (2 mentions)
Penicillin streptomycin, by HiMedia (2 mentions)
Glutamine, by HiMedia (2 mentions)
Normocin, by InvivoGen (2 mentions)
Zeocin, by InvivoGen (2 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Microgem (1 mentions)
Renca, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Euroclone (1 mentions)
Renca, by American Type Culture Collection (1 mentions)
Geneticin, by Biowest (1 mentions)
Doxycycline, by Takara Bio (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Takara Bio (1 mentions)
Vr 1558, by American Type Culture Collection (1 mentions)
Vero 76, by American Type Culture Collection (1 mentions)
19 protocols using fetal bovine serum (fbs)
1
Stem Cell Differentiation Protocols
2
Establishment and Maintenance of Renal Adenocarcinoma and Fibrosarcoma Cell Lines
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Renal adenocarcinoma cell line Renca was purchased from the American Type Culture Collection (ATCC). Tumor cells were maintained in RPMI-1640 (Microgem), supplemented with 10% fetal bovine serum (FBS; Microgem). The Renca-Luc variant was obtained by infecting Renca cells with a lentiviral vector carrying the coding sequence of synthetic firefly luciferase gene luc2 and using blasticidin (5 µg mL−1; Gibco) as a selection antibiotic. The Renca-iRFP670 is the fluorescent Renca variant stably expressing the iRFP670 fluorescent protein. The UV-2237 fibrosarcoma cell line26 (link) was maintained in DMEM (EuroClone) supplemented with 10% FBS. Stocks of the cell lines were stored frozen in liquid nitrogen and used within four weeks after thawing. Cells were routinely tested by PCR for Mycoplasma contamination.
The Lewis Lung carcinoma cell line (LLC) was obtained from the Developmental Therapeutics Program (DTP) – Division of Cancer Treatment and Diagnosis (DCTD) Tumor Repository, NCI, US. LLC was stocked frozen as in vivo-derived tumor fragments and maintained subcutaneously in C57/BL6 mice before testing.
The Lewis Lung carcinoma cell line (LLC) was obtained from the Developmental Therapeutics Program (DTP) – Division of Cancer Treatment and Diagnosis (DCTD) Tumor Repository, NCI, US. LLC was stocked frozen as in vivo-derived tumor fragments and maintained subcutaneously in C57/BL6 mice before testing.
3
HeLa Cell Culture Optimization
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HeLa cells (ATCC number CCL-2) were employed in all cellular experiments. Both bidimensional and tridimensional cultures were maintained in Dulbecco’s Modified Eagle’s Medium (Microgem Naples, Italy, TL1006) supplemented with 10% Fetal Bovine Serum (Microgem, RM10432), 2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin (Microgem) and cultured at 37 °C, 5% CO2. Bidimensional cultures were passed upon trypsin digestion every two or three days; the growth medium of 3D constructs was changed regularly, with a fresh one every two or three days.
4
Doxycycline-Induced C2C12 Differentiation
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C2C12 cells were obtained from ATCC and cultured in Dulbecco's modified Eagle Medium (DMEM) supplemented with 10% FBS (Microgem), 2 mM glutamine, 50 i.u. penicillin, and 50 μg/ml streptomycin (proliferating medium). C2C12 TET-ON D2 cells were cultured in DMEM supplemented with 10% tetracycline-free Fetal Bovine Serum (FBS) (Clontech, Mountain View, CA, USA), 100 μg/ml geneticin (Biowest, Nuaillé, France) and 0.8 μg/ml puromycin (Invitrogen, Carlsbad, CA, USA). For studies in proliferative conditions (PROL), cells were grown at 40–50% confluence and treated with the indicated amounts of doxycycline for 16, 24 or 48 h as indicated. For experiments in differentiation conditions (DIFF), cells were grown at 60%–70% confluence, treated with doxycycline and then induced to differentiate in Differentiation Medium (DMEM with 2% horse serum, insulin 10 μg/ml and transferrin 5 μg/ml). D2 expression was turned on by 2 μg/ml doxycycline (Clontech, Mountain View, CA, USA). In some experiments, TH (T3 and T4, Sigma-Aldrich S. Louis, Missouri, USA) were added in culture medium at a 30 nM final concentration or removed from the FBS by charcoal absorption (Larsen, 1972).
5
H9c2 Cell Viability Assay
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H9c2 cells were grown to confluence in Dulbecco’s modified Eagle’s Medium (DMEM; Microgem, Charlottesville, VA, USA) with 10% fetal bovine serum (FBS; Microgem) and antibiotics (25 U/mL penicillin and 25 U/mL streptomycin) under an atmosphere of 95% air/5% CO2 at 37 °C. H9c2 cells were seeded in DMEM 10% FBS at right confluence for each experiment and exposed to NSS and AU extracts (50 μg·mL−1) for 4 h. Extracts were then washed away and doxo (1 µM) or fresh medium was added for other 20 h.
6
Cell Lines and Virus Propagation for COVID-19 Research
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Cercopithecus aethiops kidney cells (Vero-76, ATCC CRL 1587) and human lung adenocarcinoma Calu-3 (ATCC HTB-55) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Vero-76 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Microtech, Naples, Italy) supplemented with 10% Fetal Bovine Serum (FBS, Microgem, Naples, Italy) and antibiotic solution (10,000 U penicillin + 10 mg streptomycin, Himedia, Mumbai, India). Calu-3 cells were grown in Eagle’s Minimum Essential Medium (EMEM, Microtech) supplemented with 10% FBS antibiotic solution. All the coronaviruses used in this study, which are (i) HCoV-229E (ATCC VR-740); (ii) HCoV-OC43 (ATCC VR-1558); and (iii) SARS-CoV-2 (clinical isolate, strain VR PV10734, kindly donated by the Lazzaro Spallanzani Hospital, Rome, Italy), were propagated in the Vero-76 cell line and their concentration was determined via plaque assay [20 (link)]. For HCoV-OC43, the viral concentration was expressed as the median tissue culture infectious dose (TCID50). All experimental work involving SARS-CoV-2 was performed in a biosafety level (BSL) 3 laboratory.
7
Cardiomyocyte Cell Culture Protocol
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Doxo was obtained from Baxter Manufacturing Spa. Radicicol and L-NAME were from Sigma-Aldrich; Merck KGaA. The H9c2 embryonic rat heart cardiomyocyte-derived cell line was purchased from the American Tissue Culture Collection (ATCC), with mycoplasma testing carried out. The H9c2 cells were grown to confluence in Dulbecco's modified Eagle's medium (DMEM; Microgem) with 10% fetal bovine serum (FBS; Microgem) and antibiotics (25 U/ml penicillin; 25 U/ml streptomycin) under an atmosphere of 95% air/ 5% CO2 at 37°C.
8
SFNV Propagation in Vero Cells
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Vero cells (ATCC CRL-1587) were grown at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Microtech, Naples, Italy) with the addition of 10% inactivated fetal bovine serum (FBS, Microgem, Naples, Italy), 2 mM glutamine, and 1X antibiotic solution (penicillin/streptomycin solution). SFNV strain UVE/SFNV/UNK/IT/30451 (acquired by the European virus archive global, EVAg) was propagated in Vero cells and titrated by median tissue culture infectious dose (TCID50). In brief, the virus was serially diluted and incubated in 96-well plates containing 2 × 105/mL adherent Vero cells seeded the previous day. Plates were incubated at 37 °C for 5 days prior evaluation of CPE via microscope and were then fixed and stained with Gram’s crystal violet solution.
9
Propagation of Viral Strains on Vero Cells
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African green monkey kidney epithelial cell lines (Vero CCL-81, Manassas, VA, USA) were acquired from the American Type Culture Collection (ATCC), cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose (Microtech, Naples, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Microgem, Naples, Italy), 2 mM L-glutamine (Microtech), and 100 IU/mL of penicillin–streptomycin solution (Himedia, Naples, Italy), and maintained at 37 °C in a humidified atmosphere with 5% CO2. All described viral strains were propagated on Vero cell lines: HSV-1 (strain SC16), HSV-1expressing the VP22-green fluorescent protein, HSV-2 (strain 333), poliovirus type 1 (PV-1 strain CHAT), human coronavirus 229E (HCoV-229E strain VR-740), and SARS-CoV-2 (strain VR-PV10734; kindly provided by Lazzaro Spallanzani Hospital, Rome, Italy) [58 (link)]. The experiments involving SARS-CoV-2 were carried out in a biosafety level 3 (BSL-3) containment laboratory (University of Salerno) according to World Health Organization (WHO) recommendations.
10
Ovarian Cancer Cell Culture Protocol
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OVCAR3 (NCI-DTP; cat. #OVCAR3, RRID:CVCL_0465) and IGROV1 (NCI-DTP; cat. #IGR-OV1, RRID:CVCL_1304) human ovarian adenocarcinoma cells were obtained from the NCI (Fredrick, MD) and were routinely cultured in RPMI-1640 medium (Microgem) supplemented with 10% FBS (Microgem) and 2 mmol/L L-glutamine (GIBCO). Stocks of cell lines, authenticated by short-tandem repeat profiling (AmpFlSTR Identifiler Plus PCR Amplification Kit; Applied Biosystems), were stored frozen in liquid nitrogen and used within 4 weeks after thawing. Cells were routinely tested for Mycoplasma contamination by PCR.
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