Computational protocol: Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

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Protocol publication

[…] The mRNA-transfected human CAR-T cells, which were stimulated with antigenic proteins for 3, 6, or 12 hours, were pulsed with BD Golgiplug (BD Biosciences) and BD Golgistop (BD Biosciences) for the last 3–6 hours of antigenic stimulation in ALyS505N-175 and then fixed with BD Cytofix/Cytoperm (BD Biosciences) overnight at 4 °C. Cells were stained with Live/Dead Fixable Aqua viability dye, fluorescent-labeled mAbs for anti-human IFN-γ (clone 4S.B3, eBioscience), anti-human TNF-α (clone MAb11, eBioscience), anti-hIL-2 (clone MQ1-17H12, eBioscience), and anti-human CD107a (hCD107a) (clone H4A3, eBioscience). The frequency of each factor-positive cell in the gated Aqua− CD8α+ cells was analyzed by FCM, as described above. The amount of IFN-γ, TNF-α, IL-2, and granzyme-B secreted from the mRNA-transfected human CAR-T cells after 24 hours stimulation in ALyS505N-0 was determined by the Human IFN-γ ELISA Set (BD Biosciences), the Human TNF ELISA Set (BD Biosciences), the Human IL-2 ELISA Kit II (BD Biosciences), and the Human Granzyme-B ELISA development kit (Mabtech, Cincinnati, OH). […]

Pipeline specifications

Software tools CAR, AQUa
Application De novo sequencing analysis
Organisms Mus musculus, Homo sapiens
Diseases Neoplasms, Nervous System Diseases, Leukemia, T-Cell