Journal:
Article Title: Annexin 2 is a Regulator of SDF-1/CXCL12 Function in the Hematopoietic Stem Cell Endosteal Niche
doi: 10.1016/j.exphem.2010.11.007
Figure Lengend Snippet: (A) In vitro binding assays between CXCL12 and annexin 2 were performed to determine binding specificity. Annexin 2 was used as a binding target for CXCL12 and non-specific binding was blocked with bovine serum albuman (BSA). Parathyroid hormone (PTH), fibronectin (FN), collagen 1a (COL1a) or anti-annexin 2 antibody was used as competitors. (B) Biotinylated CXCL12 (bCXCL12) was incubated alone or together with annexin 2. Immune precipitation (IP) was performed with an antibody to annexin 2 and the proteins were collected on protein G sepharose beads. bCXCL12 detection accomplished by Western blot using strepavidin-HRP. Control-proteins were run without IP. (C) Co-localization of CXCL12 with annexin 2 was determined by immunofluorecent staining on the frozen sections of the long bones in the wild-type (anxa2 +/+) animals. Annexin 2 positive cells were detected by anti-annexin 2 antibody (red). CXCL12 positive cells were detected by anti-CXCL12 antibody (green). DAPI stained nuclei (blue). Co-localization of CXCL12 with annexin 2 (yellow-orange) was showed on the endosteal surfaces. Original magnification at 60x. Bar = 100 microns. (D) Migration assays of LSK cells from anxa2+/+ and anxa2−/− animals were performed using dual chambers to determine the hematopoietic progenitor cell migration in response to CXCL12-annexin 2 interactions. (E) Erk and Erk phosphorylation in LSK cells were evaluated by FACS analysis to determine the signaling in response to CXCL12-annexin 2 interactions. (F) Hematopoietic progenitor colony-forming cell assays performed on 2×105 cells that migrated to CXCL12, annexin 2 or the combination. Data are presented as the mean ± standard error of the mean. *P<0.05 as determined by Student-T test.
Article Snippet: Cells were incubated first with a biotinylated anti-lineage (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], and Ter-119) antibody cocktail (Miltenyi Biotec, Auburn, CA) and an antibody cocktail of anti-CD150PE (Clone TC15-12F12.2, BioLengend, San Diego, CA), CD48FITC (Clone BCM-1, BD Pharmingen, San Diego, CA), CD41FITC (Clone MWReg30, BD Pharmingen), c-KitPE/Cy7 (Clone 2B8, eBioscience, San Diego, CA), and Sca1APC (clone D7, eBioscience) for 20 min on ice, then rinsed, and stained with anti-BIO micro beads (Miltenyi Biotec) and streptavidin-APC/Cy7 conjugated secondary antibody (BD Pharmingen) for another 20 min. Lin − cells were then enriched by magnetic separation (AutoMACS, Miltenyi Biotec), and then resuspended in 2 mg/ml 7-AAD (eBioscience) to discriminate live from dead cells.
Techniques: In Vitro, Binding Assay, Incubation, Western Blot, Staining, Migration
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