Human Coronary Artery Endothelial Cells (HCAECs) from ATCC (Manassas, VA, USA), Human Aortic Endothelial Cells (HAECs) from Thermo Fisher Scientific (Waltham, MA, USA), and Human Umbilical Vein Endothelial Cells (HUVECs) from Thermo Fisher Scientific were grown on 1.5% gelatin-coated tissue culture dishes and maintained in medium M200 (Thermo Fisher Scientific, Waltham, MA, USA) containing 2% fetal bovine serum (FBS) and growth factors (EGM-2, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C with 5% CO2. Experiments were performed in actively proliferating ECs at passages from 3 to 5. KRIT1−/− and KRIT1+/+ mouse embryonic fibroblast (MEF) cell lines [21 (link)] were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and 100 U/mL penicillin/streptomycin. Cell treatments were performed with either Tumor Necrosis Factor α (TNF-α) (10 ng/mL for 24 h) or Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) (0.5 or 1 mM for 24 h).
Egm 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EGM-2 is a compact and portable CO2 gas analyzer designed for various applications. It provides accurate measurements of CO2 concentration in the range of 0-10,000 ppm, with a fast response time. The EGM-2 employs a nondispersive infrared (NDIR) sensor technology to detect and measure CO2 levels.
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Lab products found in correlation
Egm 2, by Lonza (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ebm 2, by Lonza (2 mentions)
Purified rat anti mouse cd31, by BD (2 mentions)
Collagenase type 2, by Thermo Fisher Scientific (2 mentions)
M 450 sheep anti rat igg, by Thermo Fisher Scientific (2 mentions)
Growth factors, by Lonza (2 mentions)
Hcaec, by American Type Culture Collection (1 mentions)
M200 medium, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Corning (1 mentions)
Dnase, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Red blood cell lysis buffer, by BD (1 mentions)
Observer z1, by Zeiss (1 mentions)
Egm 2, by Merck Group (1 mentions)
6 aminocaproic acid, by Merck Group (1 mentions)
18 protocols using egm 2
1
Endothelial Cell Culture Conditions
2
Isolation and Culture of Fetal Lung Fibroblasts
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Human fetal lung fibroblasts (FLFs) were isolated from 18‐ to 20‐week‐old fetal lungs (Advanced Bioscience Resources, Alameda, CA). Tissues were finely minced and dissociated using 1 mg/ml collagenase/dispase (Roche, Basel, Switzerland, http://www.roche.com ) and 0.1 mg/ml DNase (Sigma‐Aldrich) with rotation for 45 minutes at 37°C. After washing in media containing 1% fetal bovine serum (FBS), a single‐cell suspension was generated using 100‐ and 40‐μm cell strainers. To remove red blood cells, the suspension was incubated in red blood cell lysis buffer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com ) for 15 minutes at room temperature. Cells were then plated in 6‐well tissue culture plates and cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 10% FBS (Corning). Human umbilical vein endothelial cells (HUVECs) and small airway epithelial cells (SAECs) were maintained according to the manufacturer's recommendations (Lonza, Basel, Switzerland, http://www.lonza.com ) in endothelial growth medium (EGM)‐2 (Thermo Fisher) and small airway growth medium (SAGM) (Lonza), respectively.
3
Cell Viability and Degradation in Fibrin Hydrogels
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To analyze cell viability in the HS fibrin gels, passage 3–5 AFC were dissociated and resuspended in EGM-2 (Lonza) at 4x105 cells/mL. 75 uL HS (250 mM NaCl) and PS (145 mM NaCl) fibrin gels were fabricated using the 4X AFC suspension as drops in wells of a 6-well tissue culture-treated plate (three gels per well). After gelation, 2 mL of EGM-2 was added to each well and the gels were incubated at 37°C and 5% CO2. After 1, 24, and 96 hours, EGM-2 was aspirated and cells were stained using the fluorescent LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen) according to kit instructions. Three images were captured from each gel using a Zeiss Observer.Z1 and long-distance objective (LD Plan-NEOFLUAR 20X/0,4 Ph2) and were used to count living and dead cells.
To analyze the cell-mediated degradation kinetics of HS and PS fibrin formulations, gels were fabricated with a final concentration of 1x105 AFC/mL (P3-5) and incubated in EGM-2 +/- 1 mg/mL of the plasmin inhibitor 6-aminocaproic acid (Sigma, A2504) at 37°C and 5% CO2. After 0, 7, and 14 days, gels were imaged using phase contrast (Zeiss ObserverZ.1) and wet weights were recorded.
To analyze the cell-mediated degradation kinetics of HS and PS fibrin formulations, gels were fabricated with a final concentration of 1x105 AFC/mL (P3-5) and incubated in EGM-2 +/- 1 mg/mL of the plasmin inhibitor 6-aminocaproic acid (Sigma, A2504) at 37°C and 5% CO2. After 0, 7, and 14 days, gels were imaged using phase contrast (Zeiss ObserverZ.1) and wet weights were recorded.
4
Oxidative Stress Induction in Pseudoislets
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To induce oxidative stress, the pseudoislets were incubated with 250 µM hydrogen peroxide (H2O2) diluted in phenol-free EGM-2 (PromoCell) after being washed with PBS for 2 min. After 30 min incubation in H2O2, 10 µM of the fluorogenic probe CellROX Deep Red Reagent (Invitrogen) in phenol-free EGM-2 with/without 250 µM H2O2, was added into the existing medium to a final concentration of 5 µM. Control samples were incubated with phenol-free EGM-2 for 60 min at 37°C. After 30 min of incubation with CellROX Deep Red Reagent, the pseudoislets were fixated in 4% (wt/vol) formaldehyde diluted in PBS for 20 min at room temperature and were subsequently, washed two times with PBS for 3 min.
5
Matrigel-Based Angiogenesis Assay
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One hundred μL of Matrigel (BD Biosciences) basement membrane matrix was added to 24-well plates. The plates were then incubated for 30 min to allow gel solidification. Then, 2×104 cells from 15-day HD or RD cultures were seeded onto the gel in 500 µL EGM-2 (Invitrogen). Twelve hours later, the plates were observed under a light microscope (Olympus, Tokyo, Japan). Nine representative fields were recorded and the average number of branch points was calculated by Image-Pro Plus software (Media Cybernetics, Atlanta, GA, USA).
6
Endothelial Cell Culture Protocols
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All culture media (except EBM-2 and EGM-2), L-cysteine, fibronectin, trypsin, penicillin, and streptomycin were from Invitrogen (Breda, the Netherlands). EBM-2 and EGM-2 were from Lonza (Verviers, Belgium). Epinephrine, thrombin, forskolin, 3-isobutyl-1-methylxanthine (IBMX), LY294002, Y27632, BAPTA-AM, Endothelial Cell Growth Supplement (ECGS), and anti-α-tubulin monoclonal antibody (DM1A) were from Sigma-Aldrich Chemie (Steinheim, Germany). S1P was from Avanti Polar Lipids (Alabaster, Alabama, USA). Anti-β-catenin rabbit polyclonal antibody (sc-7199) was from Santa Cruz Biotechnology (Santa Cruz, California, USA). Chemiluminescence blotting substrate and Complete Protease Inhibitor Cocktail Tablets were from Roche Diagnostics (Mannheim, Germany). All chemicals used were of analytical grade. Anti-VWF monoclonal antibody CLB-RAg20 has been described previously [29] (link). Enzyme-linked immunosorbent assays (ELISA) for VWF and VWF propeptide have been described previously [30] (link).
7
Isolation of Fibroblasts and Endothelial Cells
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Anesthetized mice were perfused with PBS, pH 7.4, to wash out erythrocytes. Dorsal skin tissue samples of 7 week-old mutant and wild type littermates were digested in 1 mg/ml collagenase type II (17101-015 Gibco® Life Technologies) in DMEM or EGM-2 (Gibco® Life Technologies) serum-free medium and P/S solution for isolation of fibroblasts or endothelial cells. Fibroblasts were cultured as described above. Dynabeads containing M-450 sheep anti-rat IgG (Life Technologies), in PBS + 0.1% BSA + 0.02% NaN3 were used to isolate endothelial cells according to manufacturer’s instructions. Purified rat anti-mouse CD31 (Pharmingen, #553369), was used to prepare anti-mouse CD31-conjugated Dynabeads by adding 5 µl of anti-CD31 antibody for each 100 µl of beads and incubation overnight at 4 °C.
To isolate CD31+ cells, 30 µl of anti-mouse CD31-coupled Dynabeads was added per 1 ml of cell suspension followed by incubation on a rotator for 1 h at room temperature, followed by separation on a magnetic separator for 1–2 min. The bound CD31+ cells (beads + cells) were cultured in EGM-2 medium, supplemented with growth factors (Lonza) and 20% FBS while the unbound fractions, CD31− supernatants, were centrifuged for 5 min at 1200 rpm and then resuspended in DMEM medium with 1% of GlutaMax and 15% FBS. CD31-positive and CD31-negative cells were plated in gelatin-coated plates.
To isolate CD31+ cells, 30 µl of anti-mouse CD31-coupled Dynabeads was added per 1 ml of cell suspension followed by incubation on a rotator for 1 h at room temperature, followed by separation on a magnetic separator for 1–2 min. The bound CD31+ cells (beads + cells) were cultured in EGM-2 medium, supplemented with growth factors (Lonza) and 20% FBS while the unbound fractions, CD31− supernatants, were centrifuged for 5 min at 1200 rpm and then resuspended in DMEM medium with 1% of GlutaMax and 15% FBS. CD31-positive and CD31-negative cells were plated in gelatin-coated plates.
8
Isolation and Culture of Primary Murine Endothelial Cells
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Primary MAECs from WT or SENP1-ecKO mice were isolated and cultured as described55 (link). HUVECs were obtained from the tissue culture core facility of the Yale Vascular Biology and Therapeutic (VBT) program and maintained in EBM-2 (Lonza) that was supplemented with 10% fetal bovine serum (Gibco), 2 mM L -glutamine, penicillin/streptomycin and the EGM-2 bullet kit in a humidified 37 °C incubator with 5% CO2 atmosphere.
HUVECs were allowed to grow to 50% confluence before GATA2 siRNA transfection. Knockdown of GATA2 was performed with Lipofectamine RNA iMAX according to the manufacturer's instructions (Life Technologies). In brief, HUVECs were transfected with GATA2 siRNA (Life Technologies) or scrambled siRNA at 20 nM in reduced serum medium (Life Technologies). The reduced serum medium was replaced with fresh complete medium at 6 h after transfection, and the cells were kept in culture for 72 h before the experiment.
For GATA2 adenoviral infection, HUVECs were infected by various GATA2 adenoviruses with polybrene (5 nM) and incubated for 48 h before further experiments.
HUVECs were allowed to grow to 50% confluence before GATA2 siRNA transfection. Knockdown of GATA2 was performed with Lipofectamine RNA iMAX according to the manufacturer's instructions (Life Technologies). In brief, HUVECs were transfected with GATA2 siRNA (Life Technologies) or scrambled siRNA at 20 nM in reduced serum medium (Life Technologies). The reduced serum medium was replaced with fresh complete medium at 6 h after transfection, and the cells were kept in culture for 72 h before the experiment.
For GATA2 adenoviral infection, HUVECs were infected by various GATA2 adenoviruses with polybrene (5 nM) and incubated for 48 h before further experiments.
9
VEGF Release by Encapsulated HUVECs
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VEGF release by human umbilical vein endothelial cells (HUVECs) was evaluated in two forms of the cultured cells alone or encapsulated within (RADA)4-SDKP. For this purpose, HUVECs were isolated from aseptic human umbilical cords that were received from Arash Hospital (Tehran, Iran) after obtaining written consent from volunteer couples, as previously described [42 (link)]. The HUVECs were cultured in EGM-2 supplemented with 10% FBS (10,270, Gibco). All in vitro experiments were performed using passages 3–6 HUVECs, and the cells were incubated at 5% CO2 and 37 °C and tested regularly for mycoplasma contamination by our laboratory. Then, 1 × 104 HUVECs were cultured alone (control) or encapsulated into the hydrogel at a final concentration of 0.25% v/w onto each well of a 48-well plate that contained the aforementioned medium for 24 or 124 h (n = 3). Next, conditioned media from the cultured cells were collected and assessed by enzyme-linked immunosorbent assay (ELISA) using a Human VEGF DuoSet ELISA DY293B-05 kit (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions.
10
Isolation and Culture of hMSCs and HUVECs
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Human bone marrow stromal cells (hMSCs) were isolated and proliferated as described previously [29] (link). Bone marrow aspirates were obtained from patients who had given written informed consent. Briefly, aspirates were cultured in minimal essential medium (α-mem; Gibco, Life Technologies) complemented with 10% v/v heat-inactivated fetal bovine serum (FBS; SA origin; Sigma-Aldrich), 100 U mL−1 of penicillin and 100 μg mL−1 of streptomycin (pen/strep; Gibco), 0.2 mM l -ascorbic acid 2-phosphate magnesium salt (ASAP; Sigma-Aldrich), and 1 ng mL−1 of basic fibroblast growth factor (bFGF; Isokine).
Human umbilical vein endothelial cells (HUVECs, ATCC) were cultured in endothelial cell growth medium (EGM-2; Gibco). While coculturing cells, a mixture of α-mem and EGM-2 medium (1:1) was used. In cases in which solely hMSCs or HUVECs were cultured, α-mem and EGM-2 medium were used, respectively.
Human umbilical vein endothelial cells (HUVECs, ATCC) were cultured in endothelial cell growth medium (EGM-2; Gibco). While coculturing cells, a mixture of α-mem and EGM-2 medium (1:1) was used. In cases in which solely hMSCs or HUVECs were cultured, α-mem and EGM-2 medium were used, respectively.
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