Primary BMVECs were isolated from E18 PAE and SAC cortices (pooling all cortices from each dam) following removal of the meninges using the Magnetic Activated Cell Sorting (MACS®) system with microbeads for negative selection of CD45+ cells and positive selection of CD31+ cells (Miltenyi Biotec). Immortalized bEnd.3 murine BMVECs were purchased from Sigma-Aldrich and were grown in Dulbecco’s Modified Eagle Medium (DMEM, Corning) supplemented with 10% fetal bovine serum (Neuromics) and 1% Penicillin–Streptomycin (Thermo Fisher Scientific). Downregulation or overexpression of miR-150-5p was achieved by transfecting bEnd.3 BMVECs with 50 nM miR-150-5p LNA™ mimics miR-150-5p LNA™ inhibitors, negative control mimics, or negative control inhibitors (Qiagen). Overexpression of Vezf1 was achieved by transfecting bEnd.3 BMVECs with 130 ng (96-well plate) or 4 μg (6-well plate) of pCMV6 Vezf1 (Origene) or pcDNA3.1 control vector (Thermo Fisher Scientific). Transfections were performed using antibiotic-free media using Lipofectamine™ 2000 according to the manufacturer’s instructions (Thermo Fisher Scientific).
Fetal bovine serum (fbs)
Manufactured by Neuromics
Sourced in United States
Fetal Bovine Serum (FBS) is a widely used supplement for cell culture media. FBS is derived from the blood of bovine fetuses and contains a complex mixture of proteins, growth factors, and other components that support the growth and proliferation of a variety of cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sucrose, by Merck Group (2 mentions)
Cd31 cells, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin l glutamine, by Thermo Fisher Scientific (1 mentions)
D luciferin, by Abcam (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Ir783, by Merck Group (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Bodipy 581 591 c11 c11 bodipy, by Thermo Fisher Scientific (1 mentions)
12 protocols using fetal bovine serum (fbs)
1
Isolation and Transfection of Brain Microvascular Endothelial Cells
2
Isolation and Characterization of Stem Cells
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Cortical bone-derived stem cell culture: cortical bone-derived stem cells were isolated from the tibias and femurs of C57BL/6 mice, as previously described [13 (link),21 (link)]. Briefly, tibias and femurs were flushed to remove all the bone marrow and then digested in collagenase at 37 °C. The digested cells were washed and plated in CBSC media until colonies of CBSCs appeared. The cells were characterized for CBSC markers and expanded for experiments.
Mesenchymal stem cell culture: the femur and tibia of C57BL/6J mice ranging from 2 to 6 months of age were isolated. The epiphysis of each were removed and each bone cavity was flushed with pre-warmed, and the complete culture medium for bone marrow extracted. Extracted marrow medium was passed through a 70 μM filter and remaining cell suspension was cultured in DMEM (Corning) containing 20% fetal bovine serum (Neuromics, Edina, Min, USA) and 1% penicillin streptomycin/L-glutamine (Gibco Life Technologies, Waltham, MA, USA). MSCs were isolated from other bone marrow cells via two passages of plastic adherence. Remaining cells were expanded and cryopreserved as previously described [40 (link)].
Cardiac-derived stem cells culture: cardiac-derived stem cells RNA and protein were generously gifted to us from Dr. Mohsin Khan’s laboratory.
Mesenchymal stem cell culture: the femur and tibia of C57BL/6J mice ranging from 2 to 6 months of age were isolated. The epiphysis of each were removed and each bone cavity was flushed with pre-warmed, and the complete culture medium for bone marrow extracted. Extracted marrow medium was passed through a 70 μM filter and remaining cell suspension was cultured in DMEM (Corning) containing 20% fetal bovine serum (Neuromics, Edina, Min, USA) and 1% penicillin streptomycin/L-glutamine (Gibco Life Technologies, Waltham, MA, USA). MSCs were isolated from other bone marrow cells via two passages of plastic adherence. Remaining cells were expanded and cryopreserved as previously described [40 (link)].
Cardiac-derived stem cells culture: cardiac-derived stem cells RNA and protein were generously gifted to us from Dr. Mohsin Khan’s laboratory.
3
Ovarian Cancer Cell Culture Conditions
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Ovarian cell lines SK-OV-3, OVCAR-4, OVCAR-8, and A2780 were cultured in RPMI-1640 (Gibco) containing 10% Fetal Bovine Serum (Neuromics, Edina, MN), L-glutamine and 1% penicillin-streptomycin in a humidified incubator (5% CO2) at 37°C. Quinacrine, z-VAD-fmk, and cycloheximide (CHX) were obtained from Millipore Sigma (St. Louis, MO). D-Luciferin, potassium salt, was purchase from BioVision (Milpitas, CA).
4
Isolation and Culture of hBMSCs
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Primary hBMSCs (Passage 0) isolated from two donors, which are termed hBMSCs-1 and hBMSCs-2 in this study, were purchased from Obatala Sciences (New Orleans, LA, USA). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Purchasing and using human stem cells for research have been approved by the IRB Office of Louisiana State University (IRB office ID: IORG0000106). Written informed consent is not required as no patients involved in this study. The cells were cultured in T-75 flasks with a medium consisting of MEM-α (Corning) plus 20% FBS (Neuromics) and 1% Penicillin-Streptomycin (Gibco) in 5% CO2 at 37 °C. The culture medium was changed every four days. Cells were passaged at 80–90% confluence at a 1:3 ratio, and different passages were obtained and cryopreserved in liquid N2. Passages 4 and 5 cells were used for the experiments.
5
Multifaceted Optical Imaging Reagents
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IR-783 was purchased from Sigma-Aldrich. IR-12N3 was purchased from Nirmidas Biotech Co. ICG (modified) for human injection was purchased from Dandong Yichuang Pharmaceutical Co. Ltd. PBS was purchased from HyClone. Erbitux (cetuximab) (2 mg/ml) was purchased from ImClone Systems Inc. BSA was purchased from Sigma-Aldrich. FBS was purchased from Neuromics. Sucrose was purchased from Sigma-Aldrich.
6
Culturing Human Dental Pulp Stem Cells
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The human DPSCs were purchased from Lonza (Catalog #PT-5025, Lonza, OR). DPSCs were cultured in alpha-modified minimum essential medium α-MEM (Fischer Scientific, IL) containing 10% fetal bovine serum (FBS) (Neuromics, MN), 1% antibiotics (penicillin-streptomycin), and glutamate solution. Passages from two to six were used in experiments.
7
Stimulation of Murine Immune Cells
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The RAW264.7 macrophage cell line was incubated in Dulbecco’s Modified Eagle’s Medium (Gibco BRL, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS, Neuromics, Edina, MN, USA), streptomycin (100 μg/mL)/penicillin (100 U/mL, Sigma-Aldrich Co, St. Louis, MO, USA) at 37 °C under humidified air with 5% CO2. Isolated spleens from BALB/c mice (Dae-Han Experimental Animal Center, Eumseong, Republic of Korea) were gently pressed and then forced through a 40 µm cell strainer in RPMI-1640 media (Gibco BRL, Grand Island, NY, USA). YAC-1 lymphoma cells were incubated in RPMI-1640 media supplemented with 10% FBS. RAW264.7 macrophage cells, splenocytes, and YAC-1 cells were incubated with SBT (1, 10, and 100 µg/mL), Stig (1 µg/mL), or lipopolysaccharide (LPS, 10 ng/mL, Sigma-Aldrich Co., St. Louis, MO, USA).
8
FFA-Induced Steatosis in Liver Cells
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Two human liver cell lines (Huh-7 and HepG2) were used for the study and they were obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China). Cells were cultured in the medium composed of Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, USA) plus 10% fetal bovine serum (FBS, Neuromics, USA) and 1% penicillin-streptomycin (Gibco, USA). All cells were grown in the standard cell incubator under standard conditions (5% CO2 and 37 °C). Cells were tested without contamination with mycoplasma. To induce free fatty acid (FFA) overloading, cells were grown up to 70~80% confluence and then treated with a mixture of long-chain FFA (oleic acid:palmitic acid = 2:1) at the concentration of 1 mM for 24 h.
9
Caco-2 Intestinal Cell Differentiation and LPS Induction
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Human Caco-2/15 intestinal cells (kindly provided by Dr. Jean-François Beaulieu (Université de Sherbrooke, Sherbrooke, Canada) were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS, Neuromics), 10 mM HEPES, 1X GlutaMAX, and 100 U/mL Penicillin/Streptomycin. Cell lines were grown until confluence, split, and plated 2 × 105 cell/well for differentiation for 21 days to induce the enterocyte phenotype. On the day of the experiment (after 21 days of differentiation), cells were treated for 24 or 48 h with media containing compounds at the indicated concentrations. DMSO controls were at 0.1%. For the LPS induction, 24 h prior to the experiment, cells were treated with 10ug/mL of LPS (Sigma) before being incubated for 24 h with the indicated compounds at the indicated concentrations. After incubation, media was removed, cells were washed with 1X PBS and frozen for RNA extraction as above.
10
Ferroptosis Induction and Detection
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Fetal bovine serum (FBS) was from Neuromics (Edina, MN). Ferroptosis inducers and inhibitors were from Cayman Chemical (Ann Arbor, MI). BODIPY™ 581/591 C11 (C11BODIPY) was from ThermoFisher Scientific (Waltham, MA). Most of the other chemicals used were from Sigma-Aldrich (St. Louis, MO).
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