All human cell lines used in this study were maintained at 37°C with 5% CO2. OCI-LY1 cells were cultured in IMDM (Gibco) supplemented with 10% FetalPlex serum (Gemini) 1% L-Glutamine (Corning), and 1% Penicillin Streptomycin (Gibco). NU-DUL1 and SU-DHL-4 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FetalPlex serum (Gemini) 1% L-Glutamine (Corning), and 1% Penicillin Streptomycin (Gibco). OCI-LY7 cells were cultured in IMDM (Gibco) supplemented with 10% heat inactivated FBS (R&D Systems) 1% L-Glutamine (Corning), and 1% Penicillin Streptomycin (Gibco). 293FTs were a gift from the Ethan Lee lab and were cultured in DMEM (Corning) supplemented with 10% heat inactivated FBS (R&D Systems), 1% L-Glutamine (Corning), and 0.5% Penicillin Streptomycin (Gibco). S2 cells were cultured in Schneider’s media supplemented with 10% heat inactivated FBS (R&D Systems) and 1% Penicillin Streptomycin (Gibco) in the dark at room temperature (∼22°C).
Fetal bovine serum (fbs)
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Morocco, Israel
Fetal Bovine Serum (FBS) is a common cell culture supplement derived from the blood of bovine fetuses. It is widely used as a growth factor-rich additive to cell culture media to support the proliferation and maintenance of a variety of cell types in vitro.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (46 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (35 mentions)
Penicillin, by Thermo Fisher Scientific (25 mentions)
Streptomycin, by Thermo Fisher Scientific (22 mentions)
Dulbecco modified eagle medium (dmem), by Corning (17 mentions)
L glutamine, by Thermo Fisher Scientific (17 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (17 mentions)
Glutamax, by Thermo Fisher Scientific (13 mentions)
M csf, by R&D Systems (10 mentions)
Rpmi 1640, by Thermo Fisher Scientific (9 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (9 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (9 mentions)
L glutamine, by Corning (8 mentions)
Fetal bovine serum (fbs), by Merck Group (8 mentions)
Rankl, by R&D Systems (7 mentions)
α mem, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Corning (7 mentions)
Dulbecco modified eagle medium (dmem), by R&D Systems (7 mentions)
Dmem f12, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Merck Group (6 mentions)
272 protocols using fetal bovine serum (fbs)
1
Cell Culture Conditions Across Cell Lines
2
Salmonella Growth Modulation by Siderophores
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Salmonella growth curves in DMEM/F12 were performed as previously described 26 . Briefly, Salmonella WT BL1, iroN BL22, iroN fhuA BL52 and iroN fhuE BL51 were grown in Nutrient Broth (NB) supplemented with dipyridyl (NBD; per liter: 3g beef extract, 5g peptone, 0.2 mM dipyridyl) overnight at 37°C. 1x10 3 cells of Salmonella were inoculated into tissue culture medium (DMEM/F12) supplemented with 10% fetal bovine serum (FBS, Corning), DMEM/F12 + 10% FBS + human lipocalin-2 (100 ng/ml; R&D Systems), DMEM/F12 + 10% FBS + human lipocalin-2 (100 ng/ml) + ferrichrome (100 nM), or DMEM/F12 + 10% FBS + human lipocalin-2 (100 ng/ml) + coprogen (100 nM). CFU were enumerated by plating serial dilution 2, 5 and 8 h after inoculation.
For growth curves without human lipocalin-2, the strains used were Salmonella WT BL1, iroN fepA BL134 and iroN fepA fhuA fhuE BL137. Growth curves were performed as described above without the addition of lipocalin-2. Deferoxamine and rhodotorulic acid were added at a final concentration of 100 ng/ml.
For growth curves with WT Salmonella supplemented with fungal siderophores, Salmonella WT BL1 was grown in minimal medium overnight at 37°C. 1x10 3 cells of Salmonella were inoculated into minimal medium alone, minimal medium + ferrichrome (100nM), or minimal medium + coprogen (100nM). CFU were enumerated by plating serial dilution every hour for 7h.
For growth curves without human lipocalin-2, the strains used were Salmonella WT BL1, iroN fepA BL134 and iroN fepA fhuA fhuE BL137. Growth curves were performed as described above without the addition of lipocalin-2. Deferoxamine and rhodotorulic acid were added at a final concentration of 100 ng/ml.
For growth curves with WT Salmonella supplemented with fungal siderophores, Salmonella WT BL1 was grown in minimal medium overnight at 37°C. 1x10 3 cells of Salmonella were inoculated into minimal medium alone, minimal medium + ferrichrome (100nM), or minimal medium + coprogen (100nM). CFU were enumerated by plating serial dilution every hour for 7h.
3
SARS-CoV-2 Infection of Engineered Cell Lines
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A549-hACE2 lung carcinoma cells expressing the human ACE2 protein (Invivogen) were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS; R&D Systems), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.5 μg/mL puromycin (Sigma). Vero E6 TMPRSS2 cells were maintained in DMEM with 4.5 g/L glucose and 2 mM l -glutamine (Gibco), 10% heat-inactivated FBS (R&D Systems), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco), and 5 μg/mL blasticidin (Invivogen). SARS-CoV-2 strain 2019n-CoV/USA-WA-1/2020 (BEI; cat. no. NR-52281) was propagated on Vero TMPRSS2 cells as described previously (52 (link)). Infected cell supernatant containing virus was collected, passed through a 0.45-μM filter, and concentrated by centrifugation at 10,000 × g for 24 h at 10°C. Virus titer was determined by TCID50 assay on Vero TMPRSS2 cells (52 (link)). ΔS-VRP(G) particles were generated as described previously (28 (link)). WT SARS-CoV-2 or ΔS-VRP(G) particles were used to infect cells at 37°C for 2 h. Fresh medium was then added to the infected cultures, followed by incubation at 37°C for an additional 24 h before RNA harvest.
4
Satellite Cell Proliferation Assay
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Satellite cells were passaged once, then seeded at 6 × 104 cells per well in Cultrex-coated 24-well plates (R&D Systems) in Ham’s F10 (Gibco) growth medium supplemented with 20% FBS (R&D Systems), bFGF (BD Biosciences), and Primocin (InvivoGen). On days 1, 2, 3, and 5 post-plating, cells were incubated with 12 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Cat# 6494, Molecular Probes, Eugene, OR, USA) in phenol red-free Ham’s F10 with 20% FBS (R&D Systems) for 4 h at 37 °C. SDS-HCl was added, cells were incubated an additional 4 h at 37 °C, mixed thoroughly and 150 μL/well were transferred into a 96-well plate and absorbance was read at 570 nm. All data are the average +/− standard deviation of three technical replicates from 3 biological replicates per time point.
5
In Vitro Osteoclast Differentiation Protocol
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In vitro osteoclast differentiation was performed as previously described46 (link). Briefly, nonadherent bone marrow cells were cultured in α-MEM (Wako) with 10% FBS (Sigma-Aldorich) containing 4000 units of Leukoprol (M-CSF, JCR Pharmaceuticals) for 2 days to obtain BMMs. Subsequently, the BMMs were cultured for 3 days in the presence of 4000 units of Leukoprol and 50 ng ml−1 of RANKL (R&D systems) with 10% FBS or 10% FBS in the presence or absence of 2% mouse serum obtained from control or MPTP-injected mice. RANKL and M-CSF were used at those concentrations for all experiments, unless otherwise noted. Differentiation into osteoclasts was evaluated by counting TRAP-positive multinucleated cells (3 or more nuclei).
6
Breast Cancer Cell Line Culture Protocols
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MCF-7 cells were obtained from ATCC (Manassas, VA) and authenticated by STR DNA Fingerprinting at MD Anderson. Cells were cultured in DMEM (Corning, Tewksbury, MA) containing 10% FBS (R&D Systems, Inc., Minneapolis, MN) and 1% penicillin/streptomycin (Gibco, Waltham, MA) in a humidified 5% CO2 atmosphere at 37 °C. MDA-MB-231 cells were obtained from ATCC (Manassas, VA), and authenticated by STR DNA Fingerprinting at Arizona Research labs. They were cultured in Leibovitz’s L-15 medium (Corning, Tewksbury, MA) containing 10% FBS (R&D Systems, Inc., Minneapolis, MN) in a humidified incubator at 37 °C. HCC1937, MDA-MB-468, and T-47D cell lines were obtained from ATCC (Manassas, VA) and HCC1937/WT BRCA1 (hereafter referred to as HCC1937 +) were obtained from Dr. Jensen [38 (link)]. Cell culture conditions have been previously described [38 (link)]. Cell lines were tested for mycoplasma using the ATCC Universal Mycoplasma Detection kit (Manassas, VA).
7
Osteocyte-like Cell Culture Protocol
8
T2 Cell-Jurkat TCR Stimulation Assay
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The stimulation assay was done as previously described (62 (link)). The assay was set up in 96-well clear round bottom microplates (Corning) with a volume of 200 μl during all incubation steps. T2 cells expressing HLA-A*02:01 were plated at a density of 50,000 cells per well in Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and penicillin-streptomycin (100 U/ml) and pulsed with 100 mM of the test peptide for 3 hours at 37°C. The cells were then washed and cocultured with Jurkat cells expressing an exogenous TCR of interest (100,000 cells per well) in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and penicillin-streptomycin (100 U/ml) for 16 hours. The next day, the cells were washed with FACS buffer and stained with anti-CD3 (APC, clone OKT3) and anti-CD69 (PE, clone FN50) antibodies for 20 min at 4°C. Cells were washed, resuspended in FACS buffer, and analyzed on the Attune NxT Flow Cytometer (Thermo Fisher Scientific). The data were analyzed using FlowJo (v10) software.
9
T2 Cell-Jurkat Co-Culture Assay
10
Expansion of ihMSCs for Experimentation
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Induced pluripotent stem cell‐derived human mesenchymal stem cells were provided by Dr. Fei Liu (Texas A&M College of Medicine) and prepared as previously described.24 Cells were seeded at 500 cells per cm2 on tissue culture polystyrene and expanded in complete culture medium (CCM) containing α‐minimum essential media (Invitrogen), 10% (v/v) fetal bovine serum (FBS, R&D Systems, Atlanta Biologicals), 4 mM L‐glutamine, and 100 U/mL penicillin and streptomycin changed every 2 to 3 days. Upon reaching 70% confluence (about 15 000 cells per cm2, six population doublings), cells were exposed to 0.25% trypsin, 0.1% EDTA (Corning) at 37°C for 5 minutes. After deactivating trypsin with CCM, cells were collected by centrifugation at 500g for 5 minutes. One cycle of seeding and recovery was defined as a single expansion passage and ihMSCs from passage 3 to 6 were used for experiments.
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