Tracrrna atto 550

Specific crRNAs (0.1 mmol/L each; PD1_crRNA1, TIM3_crRNA2, LAG3_crRNA3; Integrated DNA Technologies, IDT, Coralville, IA, USA) and universal transactivating crRNA (tracrRNA-ATTO™550, 0.1 mmol/L; Integrated DNA Technologies, IDT, Coralville, IA, USA) were mixed at equimolar concentrations and heated at 95 °C for 5 min in a ThermoMixer^® (Eppendorf, Hamburg, Hamburg, Germany). The mixture was then cooled down until room temperature (RT) was reached. Precomplexing of Cas9 (30 pmol/μL, TrueCut™ Cas9 Protein v2, Thermo Fisher Scientific, Waltham, MA, USA, #A36498) endonuclease with all crRNA/tracrRNA complexes (80 pmol/μL of PD1_crRNA1/tracrRNA, TIM3_crRNA2/tracrRNA, and LAG3_crRNA3 /tracrRNA) was completed by mixing and incubating for 10 min at room temperature. crRNA/tracrRNA/Cas9 mixture was electroporated into 2.5 × 10⁵ EL4 lymphoma cell line, 4 × 10⁶ MH and OT CD8⁺ T cells by 4D-Nucleofector^® X Unit (Lonza, Basel, Switzerland). As control of the transfection (T.C.) CD8⁺ T cells were transfected with a mock crRNA/trRNA/Cas9.
For MH and OT-1 CD8⁺ T cells nucleofection was performed 1 week after CD3/CD28 mediated activation. EL4, MH, and OT-1 CD8⁺ T cells positive for ATTO™550 were sorted and EL4 positive cells were cultured in RPMI 10% FBS, while MH and OT-1 CD8⁺ T cells in TCM with added IL-7 and IL-15.