Streptavidin conjugated apc cy7

Antibodies for lineage negative (Lin⁻) selection (B220, Gr-1, TER-119, CD4 and CD8), for sorting (anti-Sca-1-PE, Anti-c-kit-APC, biotin-conjugated lineage panel antibodies (B220, Gr-1, CD3ε, TER-119) and streptavidin-conjugated APC-Cy-7) and for multilineage engraftment analysis (PE conjugated anti-B220, Thy-1.2, Mac-1 and Gr-1) along with the appropriate isotypes were purchased from BD Pharmingen. Sheep anti-rat IgG Dynabeads® were purchased from Invitrogen. Biotinylated murine anti-CD34 was purchased from eBiosciences. CD105, CD106, CD90, CD39, CD11b were from Biolegend. Murine GFP, CD45, ASMA, Vimentin, DMP-1, Runx-2, periostin, BSP and ALP antibodies were purchased from Abcam. Osteocalcin (OCN) antibody was from Takara Bio (Clontech). DSPP antibody was from Santa Cruz Biotechnology. All secondary antibodies were from Jackson ImmunoResearch Laboratories. CD45-APC for flow cytometry was purchased from eBiosciences.

To assess the immunophenotypical profile of BM-MSCs, monoclonal antibodies (clones) to the following surface antigens were used: CD73 (AD2), CD90 (5E10), CD105 (43A4E1), CD45 (HI30), CD31 (WM59), HLA-DR (G46-6), HLA-ABC (DX17), CD39 (eBioA1), conjugated to APC, PE, FITC, Biotin, V450, V500 and PE-Cy7, as well as APC-Cy7-conjugated Streptavidin were purchased from BD Biosciences (Heidelberg, Germany), Miltenyi Biotec (Bergisch Gladbach, Germany) or eBioscience (Frankfurt, Germany). 10⁵ cells were incubated with fluorochrome-conjugated antibodies for 15 min. at 4°C. BM-MSCs were washed twice, centrifuged for 10 min. at 200 × g at 4°C, resuspended in 300 μl of PBS and analysed by using a FACS Canto II (BD Biosciences). Data were analysed by using the FlowJo software (TreeStar Inc., Ashland, OR, USA).