Three different firefly luciferase-expressing mouse cancer cell lines were used: 4T1-Luc2 (mammary carcinoma), B16F10-Luc2 (cutaneous melanoma), and MOC2-Luc2 (oral squamous cell carcinoma). The 4T1-Luc2 cells were cultured in Dulbecco’s modified eagle medium (DMEM, high in glucose with sodium pyruvate and glutamine, Fisher Scientific) with 10% fetal bovine serum (Genesee Scientific) and 500 µg/mL of zeocin (InvivoGen). The B16F10-Luc2 cells cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum (Genesee Scientific). The media was supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin (Genesee Scientific). The MOC2-Luc2 cells were cultured in HyClone™ Iscove’s Modified Dulbecco’s Medium (IMDM) and Hams F12 Nutrient Mixture (Fisher Scientific) at a 2:1 mixture with 5% fetal bovine serum, 1% penicillin/streptomycin, 5 ng/mL epidermal growth factor, 400 ng/mL hydrocortisone, and 5 mg/mL insulin (MilliporeSigma). All cells were incubated at 37°C in 5% CO2 atmosphere and passaged 1:10 when 75-90% confluent. Cell lines underwent authentication via short tandem repeat analysis and PCR-based interspecies contamination testing performed by a third-party purveyor (CellCheck Mouse 19; IDEXX), and also underwent routine Mycoplasma testing.
Fetal bovine serum (fbs)
Manufactured by Genesee Scientific
Sourced in United States
Fetal bovine serum is a common cell culture supplement derived from the blood of bovine fetuses. It provides a complex mixture of proteins, growth factors, and other components that support the growth and proliferation of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Genesee Scientific (5 mentions)
Streptomycin, by Genesee Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Genesee Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Insulin, by Merck Group (2 mentions)
Rpmi medium, by Avantor (2 mentions)
Rpmi 1640, by Genesee Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Genesee Scientific (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Brusatol, by Merck Group (2 mentions)
Penicillin streptomycin, by Genesee Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Emetine, by Merck Group (2 mentions)
L glutamine, by Genesee Scientific (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
46 protocols using fetal bovine serum (fbs)
1
Characterization of Firefly Luciferase-Expressing Mouse Tumor Cell Lines
2
Culture of K562 Cells for Experiments
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K562 cells (ATCC, CCL-243) were cultured at 37°C, 5% CO2 at a density between 0.3–1 × 106cells/ml in RPMI medium (VWR 45000–396) topped up with 10% Fetal Bovine Serum (Genesee Scientific, cat: #25–514). Cells were split at a consistent interval of 3 days, when the cells reached 106 cells/ml.
3
Characterization of Human Cancer Cell Lines
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A549 (CCL-185)- collected from a 58-year old Caucasian male, it is a hypotriploid human cell line with the modal chromosome number 66 which can be found in 24% of cells, NCI-H1299 (CRL-5803)- established from a lymph node of 43-year old White male patient with lung cancer who received prior radiation therapy, MDA-MB-231(HTB-26)- obtained from a 51-year old White female, it is an aneuploid female with a modal chromosome number 64, and MDA-MB-468 (HTB-132)- isolated from a pleural effusion of a 51-year old Black woman with a metastatic breast adenocarcinoma, an aneuploid female with most chromosome counts in the hypertriploid range with a modal chromosome number 64. The A549, MDA-MB-231, and MDA-MB-468 cells were cultured in high glucose Modified Eagle Medium (DMEM) (Genesee Scientific, San Diego, CA, USA) while NCI-H1299 cells were cultured in RPMI 1640 (Genesee Scientific, San Diego, CA, USA). All media were supplemented with 10% heat-inactivated fetal bovine serum (Genesee Scientific, San Diego, CA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Genesee Scientific, San Diego, CA, USA). The cultures were incubated at 37ºC in 5% CO2/95% humidified air. In all cases, treatment with experimental compounds was done in basal medium supplemented with 5% heat-inactivated fetal bovine serum.
4
Culturing Docetaxel-Resistant Prostate Cancer PC3 Cells
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Prostate cancer cell line PC3 was acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA, Cat# ATCC-CRL-1435) and cultured in RPMI-1640 medium (Genesee Scientific, San Diego, CA, USA, Cat# 25-506), supplemented with 10% (v/v) fetal bovine serum (Genesee Scientific, San Diego, CA, USA, Cat# 25-514), penicillin/streptomycin (Fisher Scientific, Pittsburgh, PA, USA, Cat# 30-002-CI), and normocin 1G (Fisher Scientific, Cat# NC9390718). Cells were grown under 5% CO2 at 37 °C. A docetaxel-resistant PC3 cell line (PC3-DR) was developed as indicated previously [50 (link)] and maintained in the presence of 10 nM docetaxel (LC Laboratories, Woburn, MA, USA, Cat# D-1000). This PC3-DR cell line was recently shown by our group to upregulate DFS70/LEDGFp75 and members of its protein interactome and is an excellent model for evaluating the immunoreactivity of antibodies to these proteins [50 (link)]. Short tandem repeat (STR) service provided by ATCC (Cat# ATCC-135-XV) was used to authenticate the PC3 and PC3-DR cell lines. Mycoplasma testing was conducted at least twice a year using the MycoAlertTM Mycoplasma Detection Kit (Lonza, Walkersville, MD, USA, Cat# LT07-218). Cells that were found contaminated were discarded. Cell lines were grown until approximately 20 passages.
5
MACT Cell Treatment with Sulforaphane and Brusatol
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Sulforaphane (SFN) (Cat #S4441, Sigma-Aldrich, St. Louis, MO, USA) and Brusatol (BRU) (Cat #SML1868, Sigma-Aldrich) were solubilized in DMSO and used to treat cells based on our prior findings [12 (link)]. All treatments were prepared in traditional cell culture growth media composed of high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with sodium pyruvate (Cat #25-500, Genesee Scientific, San Diego, CA, USA) and 10% fetal bovine serum (Cat #F4135, Genesee Scientific). Before treatment, MACT cells were seeded in 6-well plates at a seeding density of 3 × 105 cells per well in DMEM media and grown overnight in an incubator at 37℃/5%CO2. Three replicates of MACT cells were then treated with either 10 µM SFN, 100 nM BRU, or an equivalent amount of DMSO (i.e., control) for 24 h before the isolation of RNA. Cells were cultured, and treatments were applied in 2 mL of media/well.
6
IMCD cell culture and AKT protein analysis
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IMCD cells were purchased from American Type Culture Collection and cultured in DMEM F-12 media (Gibco), supplemented with 5% fetal bovine serum (Genesee Scientific) and 1% penicillin/streptomycin (Genesee Scientific) at 37°C and 5% CO2. Experiments were conducted in six-well cell culture-treated plates (Falcon) and performed in triplicates. Once the cells reached 70% to 75% confluence, they were serum starved overnight (18 hours) and then studied with all drug treatments performed in serum-free media. After drug treatment, the cells were rinsed two times in PBS, and then 125 µL mammalian protein extraction reagent (Thermo Scientific) with phosphatase and protease inhibitors was added to each well of the six-well plate. The cells were scraped and then sonicated 3 × 5 pulses on the lowest setting. A BCA assay was performed to measure total protein, and 10 µg protein was loaded per lane. The remaining Western blot procedure was followed, as mentioned previously. The blots were probed with pAKT (1:10,000; Cat. #4060S; RRID:AB_2315049 ; Cell Signaling Technology) [34 ] made up in 5% BSA in TBST or total AKT (1:1000; Cat. #9272S; RRID:AB_329827 ; Cell Signaling Technology) [35 ] made up in 5% milk TBST and then normalized to total protein transferred.
7
Extracellular Vesicle Depletion from Serum
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Extracellular vesicles were removed from the serum used for cell culture, incorporating an approach that removes approximately 95% of FBS-derived extracellular vesicles [54 (link),55 (link)]. Briefly, fetal bovine serum (Genesee Scientific, San Diego, CA, USA) was subjected to ultracentrifugation at 100,000× g for 18 h at 4 °C. The supernatant was filtered with a 0.22 μm filter to reduce contamination and was stored at −18 °C until needed.
8
Characterization of Human Vocal Fold Fibroblasts
9
Culture of K562 Cells for Experiments
10
Cancer Cell Line Culturing and Antibody Verification
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All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231, A549, and MIAPaCa-2 cells were cultured in high glucose Modified Eagle Medium (DMEM, Genesee Scientific, San Diego, CA, USA). NCI-H1299 cells were cultured in RPMI 1640 (Genesee Scientific, San Diego, CA, USA). All media were supplemented with 10% heat-inactivated fetal bovine serum (Genesee Scientific, San Diego, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Genesee Scientific, San Diego, CA, USA). The cultures were incubated at 37 °C in 5% CO2/95% humidified air. In all cases, treatment with experimental agents was done in basal medium supplemented with 5% heat-inactivated fetal bovine serum. Antibodies specific to B-Raf (Cat. #14814), Phospho-B-Raf (Ser445) (Cat. #2696), c-Raf (Cat. #53745), Phospho-c-Raf (Ser338) (Cat. #9427), MEK1/2 (Cat. #8727), Phospho-MEK1/2 (Ser217/221) (Cat. #9154), p44/42 MAPK (Erk1/2) (Cat. #4695), RSK1/RSK2/RSK3 (Cat. #9355), Phospho-p90RSK (Ser380) (Cat. #11989), GAPDH (HRP Conjugate) (Cat. #8884), α-Actinin (HRP Conjugate) (Cat. #12413), and anti-rabbit IgG, HRP-linked Antibody (Cat. #7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).
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