Pe conjugated anti mouse igg

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).

Blood was collected 10 days after the second immunization by retro-orbital puncture, pooled in each study subgroup (5 animals per group), and the serum was prepared. IgG levels in serum samples from vaccinated and control mice were assessed using the Mouse IgG total ELISA Ready-SET-Go! Kit (eBioscience, San Diego, CA, USA). Analyses were performed according to the manufacturer’s instructions. Microplates were read at 450 nm with a 96-well ELISA plate reader (ELX808, Bio-Tek Instruments Winooski, VT, USA). B16F10 cells were washed with PBS+0.1% sodium azide, blocked with anti-CD16/CD32 antibodies (BD Biosciences, San Jose, CA, USA), and incubated with plasma, with an equal quantity of IgG for 60 minutes on ice. The cells were washed again and incubated with PE-conjugated anti-mouse IgG (BioLegend, San Diego, CA, USA), for 30 minutes at 4°C. After washing, the cells were analyzed using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) for the binding of serum IgG.