Jurkat T Cell Transfection Protocol

Jurkat E6.1 (CD4⁺) T cells were a kind gift from the Samelson lab at the NIH and Jurkat J76 (CD8⁺) cells were a kind gift from the Acuto lab at Oxford. These cells were transfected with DNA using a NEON electroporator (Invitrogen) for the expression of proteins tagged with PAFPs. Lines of Jurkat E6.1 cells, stably expressing TCRζ–Dronpa, were available for this study from previous work^{7 (link)}. Briefly, in that work the cells were created by selection with Geneticin at 1.5 mg ml⁻¹ (G418, Invitrogen). After 2–3 weeks, the cells were sorted and single clones were grown in 96 well plates. After 3 additional weeks, the extent of protein expression was checked by flow cytometry. Cells were then evaluated using biochemistry assays, flow cytometry, confocal microscopy (510 LSCM, Zeiss) and epifluorescence, TIRF and PALM imaging, as described below.