Apc conjugated anti inos

Tumor cell suspensions were obtained after mechanical and enzymatic disruption and filtration using a mouse tumor dissociation kit (Milteniy Biotec), according to the manufacturer's instructions. To study cytokine production, cells were stimulated with a cell stimulation cocktail (ebiosciences) and Golgi Plug (BD Biosciences) for 5 h. Then, the cells were incubated with Fc Block (BD Biosciences, 553142) for 15 min at 4°C and then 30 min at RT with Zombie NIR (BioLegend, 423105). Cells were stained with various combinations of the following antibodies: V500-conjugated anti-CD45 (BD Horizon, 561487), AF488-conjugated anti CD11b (BioLegend, 101217), BV421-conjugated anti-CD206 (BioLegend, 141717), BV421-conjugated anti-CD3 (BD Horizon, 564008), PE-conjugated anti-F4/80 (BioLegend, 123110), FITC-conjugated anti-Ly6G/C (BD Pharmingen, 553127).
For intracellular staining, cells were permeabilized with Cytofix/Cytoperm and labelled with APC-conjugated anti-iNOS (ebiosciences, 17-5920) and PerCP/Cy5.5-conjugated anti-CD68 (BioLegend, 137010). Data were collected with a FACSVerse instrument and analyzed using FlowJo software version 10 (FlowJo, LLC).

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).