elispot plate reader  (Cellular Technology Ltd)

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells do not induce an allogeneic immune response in NZB/W lupus mice (A) One million WT or HIP CAR T cells were intravenously injected into fully allogeneic NZB/W lupus mice and the host immune response was quantified after 6 days. CD8 T cells were recovered from the spleen of these mice, NK cells and macrophages were taken from C57BL/6 mice, and IgM and IgG donor CAR T cell-specific antibodies (DSA) were quantified. (B) Interferon-γ (IFN-γ) ELISpot assays were performed with CD8 lymphocytes isolated from the NZB/W recipients and the injected WT or HIP CAR T cells as stimulators. Spot frequencies were automatically enumerated (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). (C) Impedance cytotoxicity assays with WT and or HIP CAR T cells as targets and isolated CD8 lymphocytes from the lupus mice as effector cells (5 animals per group, mean ± SD per time point). The cell index is normalized at time point zero hours and a drop of the curve indicates target cell killing, while a stable signal indicates target cell survival. Fluctuations in the first few hours reflect cell culture perturbations from the addition of the effector cells. (D) IgM and IgG DSAs were quantified by flow cytometry (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). The background of this assay is shown in a dashed line. (E and F) Impedance cytotoxicity assays with WT or HIP CAR T cells or MHC class I and II-deficient double knockout (DKO) cells as targets and C57BL/6 NK cells (E) or macrophages (Mac; F) as effector cells (5 animals per group, mean ± SD per time point).

Article Snippet: One hundred thousand stimulator cells were incubated with one million recipient responder splenocytes for 24 h and IFN-γ spot frequencies were enumerated using an Elispot plate reader (AID Diagnostika GmbH, Strassburg, Germany).

Techniques: Injection, Enzyme-linked Immunospot, Isolation, MANN-WHITNEY, Cell Culture, Flow Cytometry, Double Knockout