Pe conjugated anti tnf α

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).

The following mouse-specifc mAbs were used: Pe-cy7-conjugated anti-F4/80, PE-conjugated anti-CD206, APC-conjugated anti-TNF-α (all from Biolegend, USA), APC-conjugated anti-Tim-3, FITC-conjugated anti-CD80, APC-conjugated anti-iNOS (all from eBioscience, USA), PE-conjugated anti-CD86, APC-conjugated anti-IL-10 (all from BD, USA), and APC-conjugated anti-Arg-I (RD, USA).
The following human-specific (mAbs) were used: Pe-cy7-conjugated anti-CD14, APC-conjugated anti-Tim-3, and PE-conjugated anti-TNF-α were purchased from eBioscience; FITC-conjugated anti-CD206, FITC-conjugated anti-CD163, FITC-conjugated anti-CD209, PE-conjugated anti-CD80, PE-conjugated anti-CD86, and PE-conjugated anti-IL-10 were purchased from BD company.
The mice decidual lymphocytes or human decidual macrophages were incubated with corresponding mAbs at 4°C in the dark for 30 min and were then washed once; intracellular cytokine and enzyme staining were performed after cellular fixation and permeabilization as previously described (13 (link)). Analysis was performed with a FACScanto^TM II instrument (Becton Dickinson, USA).