Pe cy7 conjugated anti f4 80

Macrophages were trypsinized and Fc blocking (eBioscience 14–0161) was performed before staining with PE-Cy7-conjugatedanti-F4/80 (0.01 g/L, BioLegend 123114) or FITC-conjugated anti-CD11b (0.01 g/L, BD Pharmingen 553310). For negative control, cells were stained with the corresponding isotype control antibodies (BioLegend 400522,0.01 g/L and BD Pharmingen 553988, 0.01 g/L, respectively). Cells were analyzed using a LSRII Fortessa flow cytometer. Results were analyzed using BD Diva and FlowJo software.

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).