ESCs were directed to differentiate into cardiomyocytes as described previously15 (link). Briefly, cells were depleted from the feeder layer with a standard technique and aggregated into EBs using the hanging drop method. Next, EBs were dissociated and cultured at a density of 100,000 cells/ml for two days in serum-free media (3 parts Iscove’s Modification of DMEM (IMDM) (Cellgro): 1 part Ham’s F12 (Gibco), 0.05% bovine serum albumin (BSA), 2 mM GlutaMax (Gibco), B27 supplement (Gibco), N2 supplement (Gibco) supplemented with 50 mg/ml ascorbic acid, and 4.5×10−4 M monothioglycerol (Sigma)). Around 48 hours later, EBs were dissociated and re-aggregated in the presence of hVEGF (5 ng/mL), Activin A (5 ng/ml), and hBMP4 (0.25 ng/ml) (all from R&D Systems). EBs were further dissociated and replated at 500,000 cells/well in a 24 well plate in StemPro-34 (Gibco) supplemented with 5 ng/mL hVEGF, 10 ng/mL human basic FGF, and 25 ng/mL FGF10 (all from R&D Systems).
Hvegf
Manufactured by R&D Systems
Sourced in United States
HVEGF is a recombinant human Vascular Endothelial Growth Factor (VEGF) protein. It is a homodimeric glycoprotein that plays a key role in angiogenesis and vasculogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Hbmp4, by R&D Systems (1 mentions)
Stempro 34, by Thermo Fisher Scientific (1 mentions)
Human basic fgf, by R&D Systems (1 mentions)
Fgf10, by R&D Systems (1 mentions)
Monothioglycerol, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Activin a, by R&D Systems (1 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Mini beadbeater 24, by Biospec (1 mentions)
Methocel, by Merck Group (1 mentions)
Th4 200 microscope, by Olympus (1 mentions)
Mil 3, by R&D Systems (1 mentions)
L glutamine, by Scharlab (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
5 protocols using hvegf
1
Differentiation of ESCs into Cardiomyocytes
2
Quantifying Secreted Proteins via ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cell supernatants or cell lysates were prepared and subjected to hIL-8, hVEGF, and hHGF ELISA (R&D systems, MN) following the manufacturer’s protocol. To prepare cell lysates, cultured cells were harvested and mechanistically homogenized using Mini-Beadbeater-24 (Biospec Products, OK) and total proteins were extracted in RIPA lysis buffer (Sigma Aldrich, MO) containing a protease and phosphatase inhibitor cocktail (Abcam, Cambridge, UK). Samples were centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatants containing total protein were used for ELISA.
3
VEGF Secretion Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 5 days of in vitro culture, supernatants (7.7 ± 0.2 mL) were collected as previously described and the concentration of released human VEGF was measured by ELISA kit (hVEGF, R&D Systems) according to manufacturer’s instructions. Three donors were assessed in triplicate for each experimental group. Data are expressed as pg of protein normalized to the total amount of DNA for each relative construct.
4
Spheroid-Based Angiogenesis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVEC and BP were mixed 1:1 in Endopan 3 containing 20% Methocel (Sigma). Twenty-five microliters cell suspension drops were pipetted on non-adherent plastic plates37 (link). Subsequently, plates were turned upside-down to form the so-called hanging drops in which spheroids are contained. Plates were incubated for 24 h at 37 °C and spheroids were collected by washing the plates with 10% FCS/PBS. Spheroids were centrifuged at 200g for 5 min and resuspended in 20% FCS and 80% Methocel. The collagen mix was prepared on ice using Collagen type I (isolated from rat tail tendons), Medium 199 and NaOH (1 M) in an 8:1:1 ratio. Additionally, 1 × HEPES buffer was added to the mix to adjust the pH. The collagen solution was mixed with the spheroid solution in a 1:1 ratio and transferred to a 24-well plate. For polymerization, gels were incubated for 30 min at 37 °C and then stimulated with 50 ng ml−1 hVEGF (R&D) in basal EC medium. The assay was stopped after 24 h of incubation at 37 °C by adding 1 ml 10% PFA per well. Pictures of ten spheroids per gel were taken on an Olympus TH4–200 microscope and average sprout length was measured with Fiji.
5
Establishing Ba/F3-VEGFR2 Cell Line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Ba/F3-VEGFR2 cells were a kind gift from K. Alitalo (Helsinki, Finland) and were licensed by the Ludwig Institute for Cancer Research, New York, NY. The generation of these cells is described elsewhere (Stacker et al, 1999 (link); Makinen et al, 2001 (link)). In brief, parental mIL-3R+ Ba/F3 cells were transfected with a receptor chimera consisting of the extracellular domain of VEGFR2 and the transmembrane and cytoplasmic domain of mouse erythropoietin receptor (mEpoR). This resulted in a cell line dependent on human VEGF (hVEGF; R&D Systems, Minneapolis, USA) or mouse IL-3 (mIL3; R&D systems) for proliferation and survival. A graphical representation of these cells is shown in Figure 1 . Ba/F3-VEGFR2 cells were maintained as suspension cultures in DMEM (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated foetal bovine serum (FBS; BioWest SAS, Nuaillé, France), Penicillin-Streptomycin (Lonza), L-glutamine (2 mM; Scharlab S.L., Barcelona, Spain), mIL3 (4 ng ml−1), zeocin (500 μg ml−1; Invitrogen, California, USA) in an atmosphere of 5% CO2 at 37 °C.
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!