Cells were stained with a panel of antibodies in PBS containing 2% FBS, 1% penicillin-streptomycin, and 1 mM EDTA. Tumor cells from primary tumor samples were identified by gating out leukocytes, endothelial, and stromal cells expressing human lineage markers: CD2 (RPA-2.10), CD3 (UCHT1), CD18 (6.7), CD31 (WM59), CD45 (HI30), and CD64 (10.1). Tumor cells from xenografts passaged in mice were identified by gating out stromal cells expressing mouse lineage markers: H-2Kd (SF1–1.1), H-2Kb (AF6–88.5) and muCD45 (30–F11). Anti-CD31 was obtained from eBioscience, and all other antibodies were obtained from BD Pharmingen. All lineage antibodies were biotinylated or directly conjugated to PE/Cy5. Labeled cells were then washed and stained with streptavidin-PE/Cy5 (BioLegend). DAPI was obtained from Invitrogen.
After gating out leukocytes, endothelial, stromal, and non-viable cells, the tumor cells were profiled for expression of TLR2. Anti-TLR2 (T2.5) was obtained from eBioscience. Cells were analyzed on a BD LSRFortessa. Events collected were analyzed using FlowJo Version 9.6.4 software (Tree Star).
After gating out leukocytes, endothelial, stromal, and non-viable cells, the tumor cells were profiled for expression of TLR2. Anti-TLR2 (T2.5) was obtained from eBioscience. Cells were analyzed on a BD LSRFortessa. Events collected were analyzed using FlowJo Version 9.6.4 software (Tree Star).