W view gemini imaging splitting optics

TCR signaling was activated by using CD3/CD28 antibody clusters^{47 (link)} (Fig. 4) or anti-TCR^{4 (link)} (clone C305, Sigma-Aldrich, Fig. 7) as described previously. Briefly, biotin-conjugated IgGs were mixed with CD3 antibody (10 μg/ml) and CD28 antibody (5 μg/ml) and were further clustered with streptavidin. Then the antibody clusters were used to stimulate the T cells cultured on nonspecific immunoglobulin G (IgG,10 μg/ml) coated glass-bottom dish. For co-expression of biosensor and CAR molecule, the wild-type or ITAM mutated (XX3) 1928ζ CAR was linked downstream of the gene of biosensor by a P2A linker in the lentiviral construct. The CD19 CAR-T cells expressing biosensors were generated by lentivirus transduction of Jurkat T cells. For imaging of CAR signaling upon antigen stimulation, CAR-T cells were dropped on the glass-bottom dishes that have been coated with the NIH-3T3 cells expressing CD19 antigens. The time-lapse fluorescence images were taken with a Nikon Eclipse Ti inverted microscope at an interval of 30 s. The W-VIEW GEMINI imaging splitting optics (Hamamatsu, Japan) with an iXon Ultra 897 camera was used to capture the ECFP (a 474/40 nm emission filter) and FRET (a 535/25 nm emission filter) fluorescent signals simultaneously. During Imaging, the cells were maintained with 5% CO₂ at 37 °C using the Tokai Hit ST Series Stage Top Incubator (Tokai Hit, Japan).

Time-lapse imaging was performed with a Nikon Eclipse Ti inverted microscope. A Tokai Hit ST Series Stage Top Incubator was used to maintain a 5% CO₂ at 37°C for cells during imaging. The W-VIEW GEMINI imaging splitting optics (Hamamatsu Photonics, Hamamatsu, Japan) with a 438/29 nm excitation filter, a 474/40 nm emission filter, a 535/25 nm emission filter, an iXon Ultra 897 EMCCD camera (Andor Technology, Belfast, United Kingdom) was used to acquire the ECFP and FRET fluorescent signals simultaneously. CAR-Jurkat T cells co-expressing ZAP70 or ERK biosensor were generated by lentivirus transduction. To monitor ZAP70 or ERK signal dynamics upon antigen stimulation, CAR-Jurkat T cells were dropped on the glass-bottom dishes coated with K562-TSHR cells. From that time on, images were acquired at an interval of 30 s for 40 min. The imaging data were analyzed by Fluocell software. The ECFP/FRET ratio of ZAP70 biosensor or the FRET/ECFP ratio of ERK biosensor for each cell was normalized before comparison.