Pe conjugated anti his

To analyze the binding of RevTMs to cancer cells and RevCAR T-cells, flow cytometry was performed as detailed before (22 (link)). To demonstrate the expression of E5B9 and E7B6 epitopes on the modified RevCAR-T cells, in-house produced anti-5B9 and anti-7B6 mAbs were included. For detection of RevTM binding to T-cells and target cancer cells PE-conjugated anti-His (Miltenyi Biotec, #130120718) and DY-649-conjugated anti-Strep-Tactin^® XT (Iba-Lifesciences, #2-1568-050) together with PE-conjugated anti-mouse IgG (BioLegend, Koblenz, Germany, #405307) were used. Additionally, commercial mAbs recognizing CEA and EpCAM (Cell Signaling Technology, Danvers, MA, USA, #2383 and #2929) together with an AlexaFluor647-conjugated anti-mouse IgG mAb (Invitrogen, Karlsruhe, Deutschland, #A-21235) were employed to evaluate the expression of the antigens on the cell surface. As negative control, a mouse IgG1 isotype control (BD Biosciences, Heidelberg, Germany, #554121) was included. Furthermore, the antigen density of cancer cells was assessed with the QIFIKIT (Agilent, Waldbronn, Germany) and PE-conjugated anti-mouse IgG mAb (BioLegend, #405307) as explained before (22 (link)). All samples were analyzed using a MACSQuant^® Analyzer and MACSQuantify Software (Miltenyi Biotec).