GXM Binding Assay with Jurkat Cells

Soluble GXM was obtained from C. gattii, as reported by Wozniak and Levitz [21 (link)], at a concentration of 200 µg/mL; it was incubated with GXMR-CAR Jurkat cells (1 × 10⁶ cells/mL) for 40 min. Cells were washed to remove unbound GXM, and murine anti-GXM monoclonal antibody (18B7 clone; Merck) was incubated with GXMR-CAR Jurkat cells for 45 min on ice. The cells were washed with cold PBS and incubated with goat anti-mouse IgG biotin-conjugated secondary antibody for 45 min. After the cells were washed, streptavidin-conjugated phycoerythrin (PE) (Thermo Fisher Scientific, Waltham, MA, USA) was added, and after 30 min, the cells were washed and analyzed using flow cytometry (Millipore Guava Easycyte Mini, Burlington, MA, USA). All the above steps were also performed with non-transduced Jurkat cells to demonstrate the absence of non-specific GXM binding. The data obtained were analyzed using the FlowJo™ software (version 10, for Windows; Ashland, OR, USA: Becton, Dickinson and Company; 2019).