One day prior to transfection, HEK-293T cells were seeded into 6-well plates or 35 mm poly-L-lysine coated glass-bottom imaging dishes (MatTek) so that the cells reach 75% confluency on the day of transfection. For all transfections performed here, a total of 2 μg of DNA per well or dish was transfected using GeneJuice (Merck) except for transfection of cells used in Fig. 4b,c , which was 1 μg of DNA per well. Transfected cells were usually analyzed 40-48 h after transfection.
In general, the main components to be transfected into HEK-TCR cells were pHR-LCK*, (U6 PylT*)4-EF1α-PylRS, pcDNA5-eRF1 (E55D) and pHR-ZAP70-mRuby2 at a DNA ratio of 2:2:2:1. Any additional components were included in the mix with a ratio of 1 while maintaining the total of 2 μg DNA per transfection. For microscopy experiments of unconjugated HEK cells, pHR-IFP2.0-CaaX was transfected in addition to the main components to identify plasma membrane. Biliverdin was added to the medium at 6 h after transfection at a final concentration of 20 μM to enhance IFP2.0 fluorescence. For microscopy experiments of HEK cells conjugated to Raji B cells, additional components of pHCM-CD45RO, pHR-CBP-CSK and pHR-ICAM-1 were transfected in addition to the core LCK* components.
In general, the main components to be transfected into HEK-TCR cells were pHR-LCK*, (U6 PylT*)4-EF1α-PylRS, pcDNA5-eRF1 (E55D) and pHR-ZAP70-mRuby2 at a DNA ratio of 2:2:2:1. Any additional components were included in the mix with a ratio of 1 while maintaining the total of 2 μg DNA per transfection. For microscopy experiments of unconjugated HEK cells, pHR-IFP2.0-CaaX was transfected in addition to the main components to identify plasma membrane. Biliverdin was added to the medium at 6 h after transfection at a final concentration of 20 μM to enhance IFP2.0 fluorescence. For microscopy experiments of HEK cells conjugated to Raji B cells, additional components of pHCM-CD45RO, pHR-CBP-CSK and pHR-ICAM-1 were transfected in addition to the core LCK* components.