Cd4 fitc cd8 fitc

To assess whether EGFRvIII/CD3 TandAb-induced activation of T-cells is dependent on the presence of target cells, primary human PBMC (4 × 10⁵/well) were cultured in the presence of increasing concentrations EGFRvIII/CD3 TandAbs in the absence of EGFRvIII-positive target cells. After 5 days incubation, proliferation was assessed in a BrdU incorporation assay as previously described (28 (link)).
T-cell activation marker induction by EGFRvIII/CD3 TandAb was analyzed by FACS: primary human T-cells were cultured for 24 h with or without 10 µg/mL of TandAb in the presence or absence of F98^EGFRvIII target cells at an E:T ratio of 1:1 before T-cells were analyzed by flow cytometry for expression of the activation markers CD25, CD137, and CD69 after staining of the cell cultures with CD4-FITC/CD8-FITC (Beckman-Coulter) in combination with CD25-PE (Beckman-Coulter), CD137-APC (BD Biosciences), or CD69-APC-Cy7 (BD Biosciences). As a control, cells were cultured in the absence of antibodies and in the absence of target cells.

During the 1st–8th post-nsPEF treatment follow-up period, the blood sample was taken from tail vein. Wwhite blood cell count was tested by an automated hematology count analyzer (Siemens, Erlangen, Germany). Liver function and cytokine were tested by automatic biochemical analyzer (Olympus AU2700, Tokyo, Japan). IgG was determined by nephelometric technique (Beckman Array 360; Beckman Coulter Instruments, Brea, U.S.A). The percentages of lymphocytes CD4/CD8 were detected by Flow-cytometry by IgG1-FITC, monoclonal fluorescent antibodies, CD4-FITC/CD8-FITC (Beckman Coulter, USA).