Anti cd16 32 clone 2.4g2

Flow cytometry staining was performed according to our previous methods (Jie et al., 2017 (link)). Briefly, isolated mononuclear cells from mice placenta were stained with fixable viability dye and purified anti-CD16/32 (clone 2.4G2) followed by surface staining of FITC-CD11b, APC-Ly6G, APC-CD3, Pacific blue-CD4, APC-780-CD8, PE-Cy7-CD122, FITC-CD45.1, and Percp-Cy5.5- CD45.2 (eBioscience). Samples were collected using LSRII FACSFortessa (Becton Dickinson) and analyzed using FlowJo software (Tree Star).

Cells were plated at 2×10⁶ cells/well and stimulated for 2 hours at 37°C with ID93 (10 μg/mL) or unstimulated. GolgiPlug (BD Biosciences) was added and the cells were incubated for an additional 8 hours at 37°C. Cells were washed and surface stained with fluorochrome-labeled antibodies to CD4 and CD8 (clone 53-6.7) (BioLegend and eBioscience) in the presence of anti-CD16/32 (clone 2.4G2) for 20 minutes. Cells were washed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 minutes. Cells were washed with Perm/Wash (BD Biosciences) and stained intracellularly with fluorochrome-labeled antibodies to CD154 (clone MR1), IFN-γ (clone XMG-1.2), IL-2 (clone JES6-5H4), TNF (clone MP6-XT22), GM-CSF (clone MP1-22E9), and IL-17A (clone TC11-18H10.1) (BioLegend and eBioscience) for 20 minutes. Cells were washed and resuspended in PBS. Up to 10⁶ events were collected on an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed with FlowJo v9. Cells were gated as singlets > lymphocytes > CD4⁺ CD8- > cytokine positive. ID93 specific response frequencies were determined by subtracting the frequency of response positives of unstimulated cells from ID93 stimulated cells in matched samples.