Streptavidin pe staining

The transduction efficiency of MUC1 CAR in mouse T cells was determined by biotinylated MUC1 core peptide binding, followed by streptavidin-PE staining (BD Biosciences). T-cell phenotypes were determined by staining for CD4-PE/Cy7, CD8-Pacific Blue, CD45-FITC, CD107a-Alexa Fluor 647, and programmed cell death protein 1 (PD-1)-APC (BioLegend). The tMUC1 expression on mouse tumor cells was assessed by TAB004 staining (provided by OncoTab Inc.) conjugated with HiLyte Fluor 647 (Dojindo Molecular Technologies, Inc.), named as TAB004-Fluor 647. To detect tMUC1 on primary tumor cells, spontaneous MMT tumors were resected and digested with Collagenase IV (Life Technologies) plus DNase I (STEMCELL Technologies) to create single-cell suspensions. Cell mixtures were cultured for 2 days and the floating cells were removed. Adherent cells were harvested for staining with TAB004-Fluor 647 (BioLegend). Dead cells were excluded by 7-AAD staining (BD Biosciences). Data were acquired on BD LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (v10).

In this study cell lines were cultured in RPMI1640 at 37°C and 5% CO₂. Staining of the cell lines with a panel of antibodies provided authentication. Mycoplasma testing was performed for all cell lines using an THP-1 reporter assay developed in our lab.⁵² (link) For surface expression of mICOS constructs a h/mICOS-APC (C398.4A, Biolegend, San Diego, CA) antibody was used. αCD19 construct expression was validated with a biotinylated Strep-tag II mAb (GenScript, NJ) followed by Streptavidin-PE staining (BD Pharmingen, San Diego, CA). Membrane bound αCD3 expression on TCS was detected with a DyLight-649-labeled goat-anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch, West Grove, PA). mICOS-L and CD19 expression were verified using mICOS-L-PE (HK5.3) and CD19-APC (HIB19) from Biolegend. For TCS exclusion in reporter assays a mCD45.2-APC (104) from Biolegend was used. LSRFortessa™ or FACSCalibur™ (BD Bioscience, Franklin Lakes, NJ) flow cytometers were used for analysis, followed by a data analysis on the FlowJo software (Tree Star, Ashland, OR).