Mouse embryos free of the deciduum were rinsed in serum-free and Hepes-buffered Dulbecco’s modified Eagle’s medium and then incubated in 2.5% pancreatic/0.25% trypsin enzyme solution at 4°C for 15 min. The embryos were microdissected to isolate the mesodermal and ectodermal layers at E7.5, as previously described (40 ). The fetal heart tube with outflow tract at E9.5 and the AGM at E11.5 were also isolated by microdissection. Digested cells from AGM tissues were purified by Dynabeads CD45 (Life Technologies) and then sorted twice into lysis buffer using CD144 antibody (BD Biosciences). Samples were snap-frozen in liquid nitrogen and pulverized before RNA extraction. The protocol was approved by the Review Board on the Ethics of Animal Experiments of Shanghai Institutes for Biological Sciences.
Cd144 antibody
Manufactured by BD
The CD144 antibody is a laboratory tool used for the detection and study of the CD144 protein, also known as VE-cadherin. It is a cell surface marker that is expressed on vascular endothelial cells and plays a role in cell-cell adhesion. The CD144 antibody can be used in various immunological techniques, such as flow cytometry and immunohistochemistry, to identify and characterize cells expressing the CD144 protein.
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2 protocols using cd144 antibody
1
Isolation of Mouse Embryonic Tissues
2
Endothelial Cell Isolation and Treatment
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Liver ECs, but not lung and heart EC, are responsive to 4-hydroxytamoxyfen-induced gene excision in vitro. Four-week-old female and male mice were used to isolate EC. Briefly, livers were cut into small pieces and incubated in a solution of 2 mg/ml collagenase I (Alfa Aesar, #J62406), 1 U/ml dispase (Stemcell Technologies, #07913), and 100 μg/ml DNase I (Roche, # 10104159001), followed by mechanical dissociation. ECs were isolated with CD144 antibody-conjugated dynabeads (CD144 antibody, BD bioscience, #555289; dynabeads, ThemoFisher Scientific, #11035). Isolated ECs were cultured in dulbecco's modified eagle medium (DMEM) (Lonza, #12709-F) with 20% fetal bovine serum (FBS; R&D Systems, #S11150H), 100 μg/ml heparin (Sigma, #H3393), and 25 μg/ml ECGF (Alfa Aesar, #J64516). For gene excision, EC were treated with 1 μM 4hydroxytamoxyfen (Cayman Chemical, #14854) for three consecutive days. Before treatment with S1P (300 nM, 30 min; Cayman Chemical, #62570), C16:0-ceramide (300 nM, 30 min; Avanti, #868516), VEGF (100 ng/mL, 2 min; Peprotech, #100-20), or Insulin (1 U/ml, 2 min), EC were cultured in DMEM with 10% Charcoal-Stripped FBS for 18 h, followed by 6 h starvation in DMEM with 0.1% Charcoal-Stripped FBS.
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