Anti mouse igg f ab 2 fragment specific antibody

The FACSCanto II instrument (BD Biosciences) was used for acquisition and FlowJo v10 (FlowJo) was used for analysis of flow cytometry data. Samples were washed with and stained in PBS (Lonza) with 1% FBS. For all experiments, known negatives (e.g. NT T-cells) served as gating controls. eBioscience Fixable viability dyes (Invitrogen) were used to exclude dead cells from analysis. T-cells were evaluated for CAR expression 5-10 days post transduction using anti-CD19 (J3-119, Beckman Coulter; SJ25C1, BD Biosciences) or anti-human IgG F(ab')₂ fragment specific antibody (Jackson ImmunoResearch) for EphA2-CAR. Anti-mouse IgG F(ab')₂ fragment specific antibody (Jackson ImmunoResearch) was used to detect HER2-CAR expression. GM18 expression was analyzed by staining with anti-CD116 (4H1, BioLegend). CAR T-cell phenotype was established using the following antibodies: CD4 (SK3, BD Biosciences), CD8 (HIT8a, BD Biosciences), CD45RA (HI100, BD Biosciences), and CCR7 (G043H7, BioLegend; 150503, BD Biosciences). The beta chain of GM18 was detected with anti-CD131 (3D7, BD Biosciences). Cell surface EphA2 expression was detected using anti-EphA2 (371805, R&D Systems).

Jurkat cells were either transduced with a lentiviral cocktail (inducible Cre and lox-stop CAR reporter, Fig. 2a) followed by the indicated HS (Fig. 2b), or transduced with the lox-stop CAR reporter lentivirus alone without HS. CAR expression was quantified by flow cytometry of CAR antibody-stained cells (anti-mouse IgG, F(ab’)₂ fragment specific antibody; Jackson ImmunoResearch, 115-606-072) 24 h after HS. Non-engineered Jurkat cells were stained with the same antibody to generate the CAR+ gate.