Anti mouse cd71

Only fresh cultured cells or tissue prepared into single cell suspensions were analyzed. The harvest and digestion of MOC1 tumors or oral mucosa into single cell suspensions for analysis was performed as previously described.²⁵ (link) Nonspecific staining was minimized by staining with CD16/32 (FcR) blocking antibodies for 10 minutes prior to the addition of primary antibodies. Anti-mouse CD71, CD45.2, CD31, PDGFR, H2-K^b, H2-D^b, CD80, CD86, Fas, FasL, PD-L1, SIINFEKL:H2-K^b, CD3, CD8, CD4, CD11c, CD11b, PDCA, F4/80, Ly6G, and Ly6C antibodies were from Biolegend, anti-mouse calreticulin was from Abcam, and anti-mouse annexin V was from eBioscience. Primary antibodies were applied for 30-60 minutes at concentrations titrated for each antibody. Dead cells were excluded via 7AAD uptake and a “fluorescence-minus-one” technique was used to validate specific staining in all antibody combinations. For flow cytometric analysis of intracellular IFNβ (primary antibody ab24324; goat anti-rat IgG secondary), treated cells were exposed to brefeldin A for the final 4 hours of incubation. Fixation, permeation and staining were achieved with the intracellular fixation and permeabilization kit (eBioscience #88-8823-88) per manufacturer recommendations. All analyses were performed on a BD FACSCanto analyzer running FACSDiva software and interpreted using FlowJo (vX10.0.7r2).