To study the binding of CFHR5, PTX3, and FH to human extracellular matrix (ECM), MaxGel diluted 1:50 in TBS was immobilized on microtiter plate wells overnight at 4°C and used for subsequent binding assays (as described above). Endothelial cell–derived ECM was prepared as described (25 (link), 27 (link)) by culturing HUVEC (American Type Culture Collection; LGC Promochem, Wesel, Germany) on gelatin-coated 96-well tissue culture plates (0.2% gelatin) in DMEM (Lonza) supplemented with 10% FCS, 1% l -glutamine, and 50 μg/ml gentamicin sulfate in a cell incubator with humidified atmosphere containing 5% CO2 for 7 d at 37°C. Cells were washed and detached from the plate by incubation in DPBS containing 10 mM EDTA at 37°C. Removal of the cells was monitored by microscopy. The cell-free ECM was washed with TBS and used immediately for binding assays as described above. The production of ECM by endothelial cells was confirmed by detecting ECM components after cell detachment, using Abs against laminin, collagen type IV, and von Willebrand factor (Sigma-Aldrich).
Von willebrand factor
Manufactured by Merck Group
Von Willebrand factor is a glycoprotein involved in hemostasis. It plays a crucial role in the initial adhesion of platelets to the site of vascular injury, thereby contributing to the formation of a stable blood clot.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (1 mentions)
Egm 2mv bulletkit, by Cambrex (1 mentions)
Laminin, by Merck Group (1 mentions)
Collagen type 4, by Merck Group (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Cxcr2, by Abcam (1 mentions)
Mip 2, by Abcam (1 mentions)
Sema7a, by Abcam (1 mentions)
Il 17a, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 protocols using von willebrand factor
1
ECM Binding Assay for CFHR5, PTX3, and FH
2
Isolation and Characterization of Endothelial Progenitor Cells
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Peripheral blood from Wistar rats and normal human umbilical cord blood were used as a source of EPCs. Approval for the use of normal human cord blood was obtained by Medical University of South Carolina Human Research (Pro00017277). EPCs were isolated by density gradient centrifugation using Histopaque‐1977 (Sigma, St. Louis, MO) and cultured in EBM‐2 with supplements (EGM2‐MV BulletKit; Clonetics, San Diego, CA), but without hydrocortisone, on human fibronectin‐coated dishes. Colony‐forming units were observed after subsequent outgrowth. Attached cells were obtained after 4 to 7 days of culture and utilized for the in vitro studies. After 3 days, nonadherent cells were removed, and adherent cells were incubated for another 24 hours before the experiments were performed. EPCs were characterized by dual staining for 1,1′‐dioctadecyl‐3,3′,3′‐tetramethylindocarbocyanine, or “Dil,” and acetyl Dil‐labeled low‐density lipoprotein and lectin as well as by expression of CD31 (BD Biosciences) and von Willebrand factor (Sigma) by immunohistochemistry.
3
ECM-Binding Assays for CFHR5, PTX3, FH
4
Immunofluorescence Analysis of Paraffin-Embedded Tissue
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Paraffin-embedded tissue sections (4-µm thick) were fixed with 4% paraformaldehyde on slides for 2 h at 37°C. The sections were washed with PBS three times and then stained with the appropriate antibody: SEMA-7A (1:1,000; cat no. ab23578; Abcam) for 12 h at 4°C. Samples were incubated at 4°C for 12 h, washed with PBS, and then incubated with a specific fluorescence-conjugated IgG (1:1,000; cat no. O-6382, Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h in a light-protected chamber at 37°C. Subsequently, sections were counterstained with Von Willebrand factor (2 µg/ml, Sigma-Aldrich; Merck KGaA) or DAPI (2 µg/ml, Sigma-Aldrich; Merck KGaA) at 37°C for 2 h and mounted. In addition, tissue sections (4-µm thick) were incubated with IL-1β (1:1,000; cat no. ab200478), IL-17A (1:1,000; cat no. ab79056), TNF-α (1:1,000; cat no. ab6671), CXCR2 (1:1,000; cat no. ab14935), MIP-2 (1:1,000; cat no. ab9950), KC (1:1,000; cat no. ab798362; all Abcam) at 4°C for 12 h. Immunofluorescence signals were detected using a laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
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