The immortalized human cerebral micro vascular EC line hCMEC/D3 (provided by Dr. Pierre-Olivier Couraud, Institut Cochin, France) was cultured according to Weksler et al. (2005) (link). Endothelial basal medium-2 (EBM-2, Lonza, Walkersville, MD, United States), supplemented with 5% fetal bovine serum (FBS, PAA Laboratories, Pasching, Austria), 10 mM HEPES (PAA Laboratories, Pasching, Austria), 1% penicillin–streptomycin (Life Technologies), 1% chemically defined lipid concentrate (Life Technologies Corporation, Paisley, United Kingdom), 1.4 mM hydrocortisone (Sigma–Aldrich, St. Louis, MO, United States), 5 mg/ml ascorbic acid (Sigma–Aldrich) and 1 ng/ml human basic fibroblast growth factor (bFGF) (Sigma–Aldrich, St. Louis, MO, United States). Cultrex Rat Collagen-I (R&D Systems, Minneapolis, MN, United States) was previously coated onto the culture dishes before cells had been seeded. Human glioblastoma U87 cell line and human embryonic kidney 293T cell line were got from Cell Bank of Chinese Academy of Sciences and maintained in Dulbecco’s modified Eagle’s medium (DMEM) of high glucose supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). All the cells were cultured at 37°C with 5% CO2 in the humid atmosphere, and the medium was changed every 48 or 72 h. EMAP-II was purchased from PeproTech (Rehovot, Israel).
Chemically defined lipid concentrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Italy, Japan, Macao
Chemically defined lipid concentrate is a cell culture supplement that provides a defined mixture of lipids essential for cell growth and proliferation. It contains a standardized blend of fatty acids, cholesterol, and other lipid components required for optimal cell culture performance.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (32 mentions)
Ascorbic acid, by Merck Group (31 mentions)
Hydrocortisone, by Merck Group (25 mentions)
Glutamax, by Thermo Fisher Scientific (19 mentions)
Ebm 2, by Lonza (14 mentions)
Monothioglycerol, by Merck Group (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Human basic fibroblast growth factor, by Merck Group (10 mentions)
Ebm 2 medium, by Lonza (9 mentions)
Hepes, by Thermo Fisher Scientific (9 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (7 mentions)
Hepes, by Merck Group (7 mentions)
Dmem f12, by Thermo Fisher Scientific (7 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (7 mentions)
Ham s f12, by Thermo Fisher Scientific (7 mentions)
Ebm 2 basal medium, by Lonza (6 mentions)
Hepes, by GE Healthcare (6 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (6 mentions)
Insulin, by Merck Group (6 mentions)
Taurine, by Merck Group (6 mentions)
100 protocols using chemically defined lipid concentrate
1
Immortalized Human Cerebral Microvascular EC Line
2
Directed Cardiac Differentiation of hiPSCs
3
Cultivation of Human Cerebral Microvascular Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Human Cerebral Microvascular Endothelial Cell Line (hCMEC/D3) was obtained from Cellutions Biosystems (CLU512, Ontario, Canada) and maintained at complete EBM-2 medium at 37°C in 5% CO2. Complete medium (final concentration) EBM-2: EBM-2 Endothelial basal medium (Lonza, #190860, Basel, Switzerland), 5% Fetal Bovine Serum (Life Technologies, #14190, Carlsbad, CA); 1% Penicillin-Streptomycin (Life Technologies, #15140–122); 1.4 μM Hydrocortisone (Sigma, #H0135, St. Louis, MO); 5 μg/mL Ascorbic acid (Sigma, #A4544); 1/100 Chemically Defined Lipid Concentrate (Life Technologies, # 11905031); 10 mM HEPES (Life Technologies, #15630–080); 1 ng/mL bFGF (Sigma, #F0291).
Inserts and flasks/Petri dishes were pre-covered with rat Collagen I lower viscosity (R&D Systems, #3443-100-01, Minneapolis, MN). For coating, rat collagen was diluted in 0.02M acetic acid to a final protein concentration of 5 μg/mL. Sufficient amount of solution was added to cover the surface of culture dish and incubate at 37°C for 1 h. This was followed by washing with PBS (Life Technologies, #1653508) three times and then replaced with culture medium. Cells were passed twice weekly and seeded on Petri dishes or flasks at a density of 25,000 cells per cm2. Three-four days after seeding on flasks or Petri dishes, cells reached confluence and can be trypsinized and used until passage 35.
Inserts and flasks/Petri dishes were pre-covered with rat Collagen I lower viscosity (R&D Systems, #3443-100-01, Minneapolis, MN). For coating, rat collagen was diluted in 0.02M acetic acid to a final protein concentration of 5 μg/mL. Sufficient amount of solution was added to cover the surface of culture dish and incubate at 37°C for 1 h. This was followed by washing with PBS (Life Technologies, #1653508) three times and then replaced with culture medium. Cells were passed twice weekly and seeded on Petri dishes or flasks at a density of 25,000 cells per cm2. Three-four days after seeding on flasks or Petri dishes, cells reached confluence and can be trypsinized and used until passage 35.
4
Anti-chicken CD8 Antibody Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LC-4 hybridoma cells, which secrete anti-chicken CD8 antibody [30 (link),31 (link)], were used as a source to produce the antibody for in vivo CD8 depletion. The hybridoma cells were initially maintained in Dulbecco’s Modified Eagle’s Media (DMEM) with 20% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) in a 37 °C incubator with 5% CO2 for three days. Later, the cells were passed and maintained in the sequentially lower concentration of FBS at 10%, 5%, and 2% for every three days. Afterwards, the cells were passed and maintained in protein-free hybridoma medium II (PFHMII) supplied with 0.2% chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA). The CELLine 1000 bioreactor flask for high-density suspension cells was used (Wheaton, IL, USA) to grow the hybridoma cells for large-scale antibody production following recommendations of the company.
5
Immortalized hCMEC/D3 Cell Culture for BBB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immortalized hCMEC/D3 cell line was donated by Dr. Couraud (INSERM, Paris). The hCMEC/D3 cells (passage 28–32) were seeded on collagen coated culture flasks (2.5-3 × 104/cm2) or glass slides (4 × 104/cm2) and maintained at 37°C with 5% CO2 exposure in EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), growth factors, antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA and HEPES (10 mM). Medium was changed every 2 days until the cells reached confluence. Monolayer integrity of hCMEC/D3 cells at confluence was confirmed by phase contrast microscopy and the expression of endothelial cell-specific phenotypic markers at cell-cell junctions, as previously described [26 (link)]. For treatment, cell monolayers were exposed to CSE concentration (5-20%) diluted from freshly prepared smoke extracts as described above. Cultures exposed to CSE-free vehicle (PBS) served as controls.
6
Cigarette Smoke Exposure on BBB Endothelium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hCMEC/D3 cells donated by Dr. Couraud (INSERM, Paris) (passage 29–30) were seeded on collagen coated cell culture flasks (2.0 × 104/cm2) and maintained at 37°C with 5% CO2 in EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), Fibroblast Growth Factor (Sigma Aldrich Inc), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA and HEPES (10 mM). Medium was changed once after 2 days and then every other day until cells formed a continuous confluent monolayer. BBB endothelial monolayers were then chronically exposed (3 times/24 h) to a final 5% CSE concentration [8 (link)] derived from freshly prepared smoke extracts diluted in EBM-2 culture media. Following CSE exposure cells were processed for RNA and protein collection. Please note that a 5% CSE final concentration (using 3R4F cigarettes as reference) yields a nicotine output of ≈100 ng/mL, which is comparable to the steady state blood nicotine concentration measured in an average chronic smoker (≈20 cigarettes/day) [8 (link), 49 (link)–51 (link)]. Experiments related to nicotine exposure as a standalone agent where performed by directly diluting nicotine in the culture media at the desired concentration (100 ng/mL).
7
Chemically Defined Culture of hPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human embryonic stem cells (HESCs; H9 line, Wicell, Madison,WI) and the human induced pluripotent stem cell (hIPSC) line BHX (Cambridge Biomedical Research Centre hIPSC Core Facility), collectively termed HPSCs, were cultured under chemically defined conditions as described previously (Brons et al., 2007 (link)). Briefly, HPSCs were cultured in a chemically defined medium (CDM) containing bovine serum albumin fraction A (CDM-BSA) supplemented with Activin A (10 ng/ml, R&D systems) and FGF2 (12 ng/ml, R&D systems) on gelatine-coated plates. CDM-BSA comprised Iscove's modified Dulbecco's medium (Gibco) plus Ham's F12 NUT-MIX (Gibco) medium in a 1:1 ratio, supplemented with Glutamax-I, BSA (5 mg/ml; Europa Bioproducts), chemically defined lipid concentrate (Life Technologies), transferrin (15 µg/ml, Roche Diagnostics), insulin (7 µg/ml, Roche Diagnostics) and monothioglycerol (450 μM, Sigma).
8
Monoclonal Antibody Production from Hybridoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lc-6 and Lc-4 hybridoma cell cultures [38 (link),39 (link),40 (link),41 (link)] secreting ani-chicken CD4 and CD8, respectively, were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), as previously described [42 (link)]. The hybridoma cells were initially maintained in DMEM with 20% fetal bovine serum (FBS) that was gradually reduced to 10%, 5%, and 2% (Thermo Fisher Scientific, Waltham, MA, USA) every three days. For antibody collection, cells were maintained in protein-free hybridoma medium II (PFHMII) with 0.2% chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA). For large scale mAb production, the Wheaton CELLine 1000 bioreactor flask cells were used following the manufacturer’s protocols (Wheaton, IL, USA).
9
Cortical Organoid Generation from PSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cortical organoids were generated as described (Kadoshima et al., 2013 (link)). Briefly, human and nonhuman primate PSCs were dissociated with Accutase (Innovative Cell Technologies), and 12,000 cells were seeded into each well of low-adhesion 96-well plates (Sumitomo Bakelite) in cortical differentiation medium (Glasgow MEM, 20% knockout serum replacement, 100 μM nonessential amino acid, 100 μM sodium pyruvate, 100 μM β-mercaptoethanol, 100 U/mL penicillin-streptomycin, 3 μM IWR1e (Millipore), and 5 μM SB431542). After 18 days, organoids were transferred to a nonadhesive 9-cm petri dish and cultured with postaggregation medium containing DMEM/F12, N2, chemically defined lipid concentrate (Life Technologies), 0.25 mg/ml fungizone (Life Technologies), and 100 U/mL penicillin-streptomycin. As the organoids were cultured for longer periods of time, further supplements were added to the postaggregation medium, including 5 μg/mL heparin (StemCell Technologies), fetal bovine serum (HyClone), 1% growth-factor-reduced Matrigel (BD Biosciences), and B27.
10
Isolation and Culture of HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested from fresh umbilical cords obtained during normal delivery of healthy neonates (according to Helsinki Protocol, Semmelweis University Institutional Review Board specifically approved this study, (permission number: SETUKEB 141/2015), and all participants provided their written informed consent to participate in this study), by collagenase digestion as described earlier.42 ,43 HUVECs were kept in gelatin-precoated flasks in MCDB131 medium (Life Technologies, Carlsbad, CA, USA) completed with 5% heat-inactivated FCS, 2 ng/ml human recombinant epidermal growth factor (R&D Systems), 1 ng/ml human recombinant basic fibroblast growth factor (Sigma), 0.3% Insulin transferrin selenium (Life Technologies), 1% chemically defined lipid concentrate (Life Technologies), 1% glutamax (Life Technologies), 1% penicillin–streptomycin antibiotics (Sigma), 5 μg/ml ascorbic acid (Sigma), 250 nM hydrocortisone (Sigma), 10 mM HEPES (Sigma), and 7.5 U/ml heparin hereinafter referred to as Comp-MCDB131. Each experiment was performed on at least 3 independent primary HUVEC cultures from different individuals.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!