The time-lapse detection of sublethal damage and CTL-target cell interactions was conducted using various imaging techniques. For the detection of fluorescent reporter and dsRed OT1 CTL interactions, frames were captured at intervals of 8-12 seconds for up to 12 hours using GCaMP6s fluorescence and 2 minutes for up to 30 hours using NLS-GFP fluorescence.
To monitor CTL conjugation with Apple-53BP1trunc-expressing target cells, transmission contrast imaging of unlabeled wt OT1 CTL and fluorescence imaging of Apple-53BP1trunc were recorded at intervals of 5-10 minutes for up to 48 hours using a Leica SP8 SMD Confocal microscope. The excitation power was limited to 0.05 mW for each excitation line (561 nm for Histone-2B-mCherry, Apple-53BP1trunc, and dsRed2; 488 nm for GCaMP6s and NLS-GFP).
To monitor Ca2+ signals in OT1 CTL and target cells, spinning-disk confocal microscopy (BD Pathway) was used during the 3D co-culture of Fura2-labeled OT1 CTL with GCaMP6s-expressing B16F10/OVA cells. Frame rates of 95 seconds were used for Fura2 (340⁄380 nm) imaging and 8 seconds for GCaMP6s excitation (488 nm). Imaging periods were limited to 1 hour to minimize phototoxicity and bleaching effects.
Viability of both CTLs and target cells during long-term imaging was ensured by verifying constant CTL migration speed, maintaining morphological integrity without cell death, and monitoring unperturbed proliferation compared to bright-field imaging. Image analysis was performed using manual or semi-automated segmentation and intensity analysis tools such as ImageJ/FIJI.
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