CD28 and neoplasm: To do so, we developed an in vitro cytotoxicity assay in which we stimulated anti-CD3 and anti-CD28-primed CD8+ T cells with control or anti-PD-1 (25 μg/mL) or isolated NETs (from 2 × 106 neutrophils/mL) or isolated NETs with DNase I (100 U/mL) for 24 h with anti-CD3 and anti-CD28, then co-incubated these stimulated CD8+ T cells with MC38 tumor cells (Figure 4E).