Fitc conjugated anti cd106

To evaluate TNF-α and CD106 expression, we isolated mononuclear cells from total bone marrow using Ficoll-Paque Plus with PE-Cy7-conjugated streptavidin antibody, followed by immunoreaction with APC-conjugated anti-c-kit, APC-Cy7-conjugated anti-Ly6A/E (Sca-1), fluorescein isothiocyanate (FITC) -conjugated anti-CD106 or FITC-conjugated anti-CD45 and phycoerythrin (PE)-conjugated anti-TNF-α (eBiosciences) after Biotin Mouse Lineage Panel staining. To evaluate proinsulin expression, mononuclear cells were fixed with BD Cytofix/CytoPerm (BD Biosciences) after reacting with PE-Cy7-conjugated streptavidin antibody, APC-conjugated anti-c-kit, APC-Cy7-conjugated anti-Ly6A/E (Sca-1), and Biotin Mouse Lineage Panel. Then a rabbit anti-insulin monoclonal antibody (Cell signaling technology) and PE-conjugated anti-rabbit IgG antibody (Cell signaling technology) were applied to mononuclear cells. To deplete dead cells, LIVE/DEAD Fixable Dead Cell Blue stain kit was used before staining. Four hours after staining, cells were analyzed using a FACS Canto II with FACS DIVA software (BD Biosciences).