Mouse igg2b pe

Samples were resuspended in FACS buffer (2% FBS, 0.1% sodium azide) and stained with the following mixtures of Biolegend antibodies: BDCA1-Percp/Cy5.5, CD14-APC, HLADR-Pacific Blue, CD3/19/56-FITC, CD123-PE/Cy7, and either FcεRIα-PE or mouse IgG_2b-PE, all at manufacturer's recommended concentrations. Some samples were stained with CD45-A700 to identify hematopoietic cells and CD203c-biotin (Biolegend, at manufacturer's recommended concentrations) followed by streptavidin-A647 (Invitrogen, at manufacturer's recommended concentration) to identify mast cells. Cells were stained with antibodies and propidium iodide (PI) (Biolegend) at 1∶400 for 15 minutes at 4 degrees, washed and spun at 1300 rpm, and resuspended in FACS buffer. At least 1×10⁶ cells were run on slow or medium speed on a BD LSRII machine and analyzed with Flowjo software.

The analyses of PD-L1 and PD-L2 expression on the CETCs were performed with an extended maintrac^® approach. For PD-L1 expression analysis we used an anti-human PD-L1 phycoerythrin (PE)-conjugated antibody (clone 29E.2A3, BioLegend, San Diego, USA) at a final concentration of 0.2 μg/ml and for PD-L2 we used an anti-human PD-L2 Alexa Fluor^® 350 conjugated antibody (clone 176611, novus biologicals, Littleton, USA) at a final concentration of 2 μg/ml. The corresponding isotypic controls for PD-L1 (Mouse IgG2b PE, BioLegend, San Diego, USA) and PD-L2 (Mouse IgG2b Alexa Fluor^® 350, Novus biologicals, Littleton, USA) were used at the same final concentration. Finally, cells were visually inspected looking for a green, red and blue surface staining, but also a well-preserved nucleus (Figure 8a, 8b). For excluding expression of PD-L1/PD-L2 on hematopoetic cells we additionally performed staining with EpCAM-FITC, PD-L1-PE/PD-L2-Alexa Fluor® 350 and CD45-Pacific blue/CD45-PE antibodies (Figure 11, 12). The results for PD-L1 and PD-L2 were calculated as percentage of total number of CETCs.