Ggest that each Angptl2 and Angptl3 ordinarily function in vivo to stimulate expansion of fetal liver, and maybe also adult, HSCs. Our identification of Angptls as development variables for mouse HSCs suggests that they could also be helpful for ex vivo expansion of human bone marrow or cord blood HSCs. If that’s the case, these things may be valuable in ex vivo expansion of those cells as part of an HSC transplantation or gene therapy protocol.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript METHODSMiceWe purchased C57 BL/6 CD45.2 and CD45.1 mice from the Jackson Laboratory or the US National Cancer Institute and maintained them at the animal facility of the Whitehead Institute for Biomedical Research. All animal experiments have been performed using the approval of the Massachusetts Institute of Technology Committee on Animal Care. Angiopoietin-like proteins We created Flag uman Angptl2, the coiled-coil domain of human Angptl2, Flag-tagged fibrinogen-like domain of human Angptl2, Flag uman Angptl4, Flag uman Fgl1 and Flaghuman Mfap4 by transient transfection of 293T cells making use of Lipofectamine 2000 (Invitrogen). Following transfection, we cultured cells CXCR3 Proteins Synonyms overnight in Iscove Modified Dulbecco Medium with ten FBS, and after that washed them with IMDM ahead of culturing them in serum-free StemSpan medium (StemCell Technologies) for one more 24 h. We harvested the conditioned medium and utilized it in experiments in Figures 1, two and 5b. We constantly employed medium from mock-transfected cells as a damaging handle. We employed serum-free conditioned medium ultured mocktransfected 293T cells for 4 h before addition of purified Angptl2 or Angptl3 inside the experiments in Figure 3. To purify Serpin B6 Proteins Formulation Flag-Angptl2 and Flag-Angptl4, we cultured the corresponding plasmidtransfected 293T cells in IMDM with ten FBS for 48 h or 72 h and collected the conditioned medium for Flag-specific affinity purification.Nat Med. Author manuscript; available in PMC 2009 November 2.Zhang et al.PagePurified mouse Angptl3 (mAngptl3) expressed by sf21 cells using a baculo-virus expression method was a gift from R D Systems. We purchased GST-hAngptl5, a fusion protein of GST and human Angptl5 (hAngptl5) and made by a cell-free wheat germ in vitro transcriptiontranslation method, from Abnova Corporation. Bacterially expressed hAngptl2 and hAngptl7 had been gifts from R D Systems. Production and purification of tagged Angptl2 and Angptl4 We constructed a fusion from the cDNA encoding human Angptl2 (ref. 15) and also a Flag peptide sequence (as Flag-hAngptl2) or with Pro100 ys330 of human IgG1 Fc sequence followed by Flag (as human FC lag uman Angptls) in the C terminus. We inserted the DNA into the pcDNA3.1 ( vector (Invitrogen) downstream in the cytomegalovirus (CMV) promoter. FlagAngptl4 was similarly constructed. We transfected plasmids into 293T cells applying Lipofectamine 2000 (Invitrogen) and collected serum-containing conditioned medium at 48 h and 72 h after transfection. We added 1 tablet/50 ml of your Complete Protease Inhibitor Cocktail (Roche), 5 g/ml phenylmethylsulfonylfluoride and one hundred mM NaCl, and applied the medium to a Flag-specific epitope immunoaffinity column (ANTI-FLAG M2 affinity Gel, Sigma), applying 500 l resin/500 ml conditioned medium. We subsequently washed the column 10 times having a total of 100 volumes of TBS (50 mM Tris, pH 7.four, 150 mM NaCl) and eluted the FlaghAngptl2 or Flag Angptl4 with 0.1 mg/ml Flag peptide (DYKDDDDK) dissolved in TBS. Cell culture We plated 20 bone marrow SP Sca-1+ CD45+ c.
