Mouse VSMCs were enzymatically isolated as described in the literature [33 (link)]. In brief, the thoracic aortas of the young and aged mice were isolated, and the adventitia and intima were removed from the vessels. The aortae were cut into 1–2-mm pieces and placed into tubes containing 2 mg/mL collagenase II (Worthington Biochemical, Lakewood, NJ, USA) at 37 °C for 3–5 h. The isolated cells were washed with and plated in complete Dulbecco’s Modified Eagle’s Medium (DMEM). To ensure the purity of the isolated cells, > 95% of the cells had to be positive for the two specific smooth muscle cell markers: smooth muscle α-actin and smooth muscle myosin heavy chain for them to be used [34 ]. Early passage VSMCs were treated with Ang II (1 μM, R&D Systems), tumor necrosis factor-α (TNF-α, 20 ng/mL, R&D Systems), or mouse MFG-E8 (250 ng/mL, R&D Systems) for 24 h prior to the subsequent experiments. Human aortic smooth muscle cells (hAoSMCs) were purchased from Lonza (Basel, Switzerland) and cultured in SmGM-2 medium (Lonza) for 24 h in either the presence or absence of human MFG-E8 (250 ng/mL, R&D systems).
Mouse mfg e8
Manufactured by R&D Systems
Sourced in China
Mouse MFG-E8 is a protein that functions as a bridge between apoptotic cells and phagocytes, promoting the engulfment of apoptotic cells. It plays a role in the clearance of apoptotic cells, which is important for tissue homeostasis and immune system regulation.
Automatically generated - may contain errors
Lab products found in correlation
Tumor necrosis factor α tnf α, by R&D Systems (1 mentions)
Ang 2, by R&D Systems (1 mentions)
Haosmc, by Lonza (1 mentions)
Smgm 2 medium, by Lonza (1 mentions)
Human mfg e8, by R&D Systems (1 mentions)
Collagenase 2, by Worthington (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Cyclin d1, by Proteintech (1 mentions)
Bca kit, by Aspen (1 mentions)
2 protocols using mouse mfg e8
1
Isolation and Characterization of Mouse Vascular Smooth Muscle Cells
2
Immunoblotting for MFG-E8, AKT, and Cyclin D1
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Total tissues or cell lysates were extracted from mouse lungs, pulmonary arteries, and human PASMCs; protein concentrations were determined using a BCA kit (Aspen, Wuhan, China). Whole lysates were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and were then transferred onto PVDF membranes, which were blocked with 5% BSA in Tris‐buffered saline containing 0.5% Tween‐20 for 1 hr at room temperature. The membranes were then incubated with primary antibodies overnight at 4°C before they were reacted with HRP‐conjugated secondary antibodies. An ECL reagent was used for chemiluminescent detection, and the membranes were analyzed using the ChemiDoc MP System (BioRad Laboratories, CA). Band intensities were quantitatively measured using ImageJ (NIH, Bethesda, MD). Primary antibodies against β‐actin (TDY Biotech Co., Beijing, China), mouse MFG‐E8 (R&D Systems, Minneapolis, MN), human MFG‐E8 (Abcam), p‐AKT, AKT (Cell Signaling Technology, Danvers, MA) and cyclin D1 (Proteintech Group Inc., Wuhan, China) were used.
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