Rtca software pro immunotherapy module

The xCELLigence real-time cell analyzer (RTCA) MP instrument (Agilent Technologies, Santa Clara, CA, USA) was used to assess CAR T cell cytotoxicity and repeat killing capacity. All assays were performed in triplicate and without the addition of exogenous cytokines. First, 30,000 LM7 cells in complete RPMI were added to each well of a 96-well E-Plate (Agilent). After LM7 cells adhered to the E-Plate for approximately 24 h and reached a cell index (relative cell impedance) plateau, 15,000 T cells in complete RPMI were added. LM7 cells alone served as negative controls. The cell index was monitored every 15 min for 3 days and normalized to the maximum cell index value immediately prior to T cell plating. Percent cytotoxicity was calculated using the RTCA Software Pro immunotherapy module (Agilent).⁵² (link) For repeat killing assays, 72 h after T cell plating, media and T cells were gently removed to avoid disrupting adherent LM7 cells and plated on 30,000 fresh LM7 cells adhered to a new 96-well E-Plate. Repeat cytolysis was assessed until T cells stopped killing, defined by no CAR T cell killing greater than 50% of LM7 target cells, or over a maximum of five total stimulations per donor.