HUVEC (Lonza) cells were transfected with lentiviral GFP and human brain derived pericytes (Sciencell) were transfected with lentiviral Red Fluorescent Protein (dsRED), according to the manufacturer’s instructions (Dharmacon). HUVEC GFP were transfected with siVEGFR1 (siFlt1) and a non-targeting siRNA referred as siSHAM (ON-TARGETplus SMARTpool, Dharmacon) using Dharmafect 1 (Dharmacon). HUVEC GFP and pericytes dsRED were co-cultured at a 5:1 ratio in co-culture medium containing with EBM-2 basal medium, 2% FCS (Invitrogen), ascorbic acid and FGF from the EGM-2 bullit kit (Lonza). Then, 0.2 µg/ml of feline/human/rhesus macaque CXCL12/SDF1α, human SCF/c-kit ligand and human IL-3 (all R&D systems) were added. Subsequently, bovine collagen type 1 (Gibco) was added to the cells (final concentration of 2.2 mg/ml, pH7.4) in a 96 wells flat bottom plate. Polymerization of the protein gel was allowed for 1 hour at 37°C, 5% CO2. The gel was then overlaid with co-culture medium. Second day, different concentrations of GrK or GrKSA solutions were added to the gel. After 3 days, images were taken using an Olympus IX51 fluorescence microscope with CellSenseDimension software. Images were processed (inverted, image size and improved contrast) using Adobe Photoshop and analyzed using AngioSys (Cellworks).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.