Cell lysates were prepared using 1X lysis buffer (Cell Signaling Technology, Danvers, MA) and supplemented with phosphatase inhibitor cocktail (phosSTOP, Roche Life sciences, Indianapolis, IN). For immunoblot analysis serum-free conditioned medium was concentrated using 10 kDa cutoff filter (YM-3 Microcon, Millipore) to load 35 μg total protein/lane. Immunoblotting was conducted as described before (1 (link),3 (link)). For ELISA neat serum-free conditioned media or 4-fold diluted lysates were used. ELISAs used in the study include: ADAM12, thrombospondin-1, VEGF, MMP-9, TIMP-2 (R & D Systems, Minneapolis, MN). Protein concentration of the lysates and conditioned medium was determined using the Bradford method (Biorad laboratories, Hercules, CA).
Thrombospondin 1
Manufactured by R&D Systems
Thrombospondin-1 is a multifunctional glycoprotein that plays a role in cellular adhesion, migration, and proliferation. It is involved in various physiological and pathological processes, including angiogenesis, inflammation, and wound healing.
Automatically generated - may contain errors
Lab products found in correlation
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Timp 2, by R&D Systems (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Mmp 9, by R&D Systems (1 mentions)
Tgf β2, by R&D Systems (1 mentions)
Tgf β3, by R&D Systems (1 mentions)
Vcam 1, by Boster Bio (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Blyscan sulphated glycosaminoglycan assay, by Biocolor (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Angiostatin, by Abcam (1 mentions)
Endostatin, by R&D Systems (1 mentions)
Tumor necrosis factor (tnf), by R&D Systems (1 mentions)
Sdf 1, by R&D Systems (1 mentions)
G csf, by R&D Systems (1 mentions)
Pdgf bb, by R&D Systems (1 mentions)
3 protocols using thrombospondin 1
1
Cell Lysate Preparation and Immunoblotting
2
Quantifying Cartilage Extracellular Matrix Proteins
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Protein levels of TIMP-1, TIMP-2, MMP-1, MMP-3, MMP-13, MMP-2, MMP-9, IL-1β, TGFβ3, TGFβ2, thrombospondin-1 (R&D Systems), TIMP-3, VCAM-1 (Boster Biological Technology), TGFβ1 (IBL International), MIA (Roche), IL-8 (Invitrogen), and aggrecan (DIAsource ImmunoAssays S.A.) in supernatants were measured on days 3 and 7 by commercially available ELISA kits according to the manufacturer's instructions. IL-6 was measured by flow cytometry using Diaclone DIAplex IL-6 kit (Gen-Probe), and glycosaminoglycans were measured by blyscan-sulphated glycosaminoglycan assay (Biocolor). ELISA data were normalized by subtracting the values of medium from the samples.
3
Age-related Changes in Circulating Stem Cells
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The peripheral blood samples were obtained from younger (18 -64 years old) and older (≥ 65 years old) healthy volunteers recruited for The Dunhill Medical Trust EPC study (DMT EPC study, NCT02980354). elsewhere (Rakkar et al. 2020) (link).
The quantities of nonhaematopoietic cells (CD45-) expressing markers for stemness (CD34), immaturity (CD133) and/or endothelial maturity (KDR) were detected in peripheral blood by flow cytometry in that fluorescein isothiocyanate (BD Biosciences, Franklin Lakes, NJ, USA), phycoeryhtrin-cyanine7-(BD Biosciences), allophycocyanin-(Miltenyi Biotech), and phycoeryhtrin-(R&D Systems) labelled antibodies were used. The changes in plasma total anti-oxidant capacity (Abcam) as well as TNF- (R&D Systems), SDF-1 (R&D Systems), G-CSF (R&D Systems), VEGF (R&D Systems), PDGF-BB (R&D Systems), endostatin (R&D Systems), angiostatin (Abcam), thrombospondin-1 (R&D Systems), and thrombospondin-2 (R&D Systems) levels were assessed using the specific ELISA kits as per the manufacturers' instructions.
The quantities of nonhaematopoietic cells (CD45-) expressing markers for stemness (CD34), immaturity (CD133) and/or endothelial maturity (KDR) were detected in peripheral blood by flow cytometry in that fluorescein isothiocyanate (BD Biosciences, Franklin Lakes, NJ, USA), phycoeryhtrin-cyanine7-(BD Biosciences), allophycocyanin-(Miltenyi Biotech), and phycoeryhtrin-(R&D Systems) labelled antibodies were used. The changes in plasma total anti-oxidant capacity (Abcam) as well as TNF- (R&D Systems), SDF-1 (R&D Systems), G-CSF (R&D Systems), VEGF (R&D Systems), PDGF-BB (R&D Systems), endostatin (R&D Systems), angiostatin (Abcam), thrombospondin-1 (R&D Systems), and thrombospondin-2 (R&D Systems) levels were assessed using the specific ELISA kits as per the manufacturers' instructions.
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