Cd8 pe clone rpa t8

The Amnis ImageStream X Mark II multispectral imaging system was used. 1×10⁶ unmanipulated CB-CD8⁺ cells and eTCR^−/− CD8⁺ T cells were washed and stained with antibodies against CD3 (clone OKT3, ref. 317306, Biolegend), CD8-PE (clone RPA-T8, ref. 301008, Biolegend), and TCR-pan-αβ (clone IP6, ref. 17-9986-42, eBioscience) to detect membrane expression. Subsequently, cells were fixed, permeabilized, and stained with anti-CD3 (clone UCHT1, ref. 300420, Biolegend) to determine cytoplasmatic expression of the coreceptor.

Transduced CD8⁺ T cells were stained for 15 min at 4°C with fluorescently labeled antibodies against CD3-Pacific Blue (clone UCHT1, ref. 558117; BD) and CD8-PE (clone RPA-T8, ref. 301008; BioLegend); Fixable Viability Dye eFluor 780 (ref. 65-0865-14; Thermo Fisher Scientific) was used to evaluate transduction efficiency. Fresh, expanded, and transduced cells were stained with Fixable Viability Dye eFluor 780 (ref. 65-0865-14; Thermo Fisher Scientific), CD62L-BV650 (clone DREG-56, ref. 2124160; Sony Biotechnology), CD25-PerCP Cy5.5 (clone BC96, ref. 2113130; Sony Biotechnology), TIM3-APC (clone 34482, ref. FAB2365A; R&D Systems [R&D]), CD3-AF700 (clone UCHT1, ref. 300424; Sony Biotechnology), CD45RA-BV421 (clone HI100, ref. 304118; BioLegend), CD45RO-BV711 (clone UCHL1, ref. 304236; BioLegend), LAG3-PE (ref. FAB2319P; R&D), CD8-PE-Cy7 (clone SK1, ref. 335822; BD), TCR Vβ21.3 (clone IG125, ref. PN IM1483; Beckman Coulter), and CD19 CAR detection reagent (ref. AB_2811310; Miltenyi Biotec). Flow cytometry analysis was performed on a BD LSRFortessa using FACSDiva, with acquisition of a fixed number of cells for each experiment. FACS data analysis was performed using FlowJo version (v.)10 . Data are presented as percent of cells and as MFI.