Trustain fcx affinity purified anti mouse cd16 32 fc block

Approximately 100 μl of blood was collected in 200 μl of ACD buffer (Becton Dickinson) and processed to remove all red blood cells prior to lymphocyte staining. Cells were treated with TruStain FcX affinity purified anti-mouse CD16/32 Fc block (BioLegend) and stained with DYKDDDDK (FLAG) Tag specific FITC-anti-Flag M2 mAb (Sigma Cat. #F4049) and PE-anti-mouse CD3e mAb (eBioscience Cat. #eBio500A2). Cell samples were analyzed on a BD FACSCaliber cytometer (BD Biosciences).

Flow cytometry was carried out to identify the presence of surface expressed 3×FLAG tagged mIR protein. Cells were treated with TruStain FcX affinity purified anti-mouse CD16/32 Fc block (BioLegend) and stained with DYKDDDDK (FLAG) Tag specific polyclonal Alexa Fluor^® 647 conjugated rabbit antibody, detected in the allophycocyanin (APC) channel (Cell Signaling Technology, cat#3916s). Cell samples were analyzed on a BD FACSCaliber cytometer (BD Biosciences).
In order to make a stable HEK-Cre+ 3×FLAG-mIR expressing cell line, the pPNTlox2-3×FLAG-mIR plasmid was linearized with Afl-II and Spe-I to remove all residual bacterial components (Fig. S1). The linearized expression cassette was transfected into HEK-Cre+ expressing cells to allow non-specific homologous recombination to occur. Cells were stained for 3×FLAG tagged mIR protein and sorted to recover eGPF and APC double positive cells. The FACS sorted double positive cells, which will be referred to as the 3×FLAGmIR cell line, were allowed to recover prior to two additional rounds of cell sorting to ensure gene integration and protein expression. Cells were sorted using the BD FACSAria IIu flow cytometer (BD).