880 inverted confocal microscope

Immunofluorescence images were acquired using either an LSM 510 or 880 inverted confocal microscope (Zeiss) with Zen software (v.2.1, Zeiss). A 63 × and 40 × plan-apochromat objective were used for immunological synapse analysis and conjugate quantification respectively. A differential interference contrast image (DIC) was acquired for assessment of cell morphology.
Slides were initially scanned for conjugates comprising a CD8⁺ cell and CD8⁻ cell to identify the correct cell pairing. For immunological synapse imaging, a single image in all colours (CD8 in 568 nm channel; synapse marker in 488 nm channel; where applicable, Gag p24 in 647 nm channel; DIC) was taken at the cell equator, followed by a Z-stack of the synapse marker channel (26 images, 0.37 Z-step μm). At least 10 conjugates per experimental condition were imaged. The number of T2-T cell conjugates were quantified by first randomly selecting regions containing T2 cells and then using the 40 × objective to acquire multiple overview images of the slide, until at least 50 target cells had been identified.

For live-cell
imaging, HEK293T cells were plated in 35 mm glass-bottom dishes (MatTek)
in phenol-free complete DMEM medium and transfected with mCherry-U2AF1
plasmid (Addgene, #84017). After 24 h, the cells were treated with
10% 1,6-hexanediol and immediately imaged on a Zeiss 880 Inverted
confocal microscope in a 37 °C humidified chamber with 5% CO₂ using a 40× oil lens.
For fixed-cell imaging,
cells were plated on cover glasses in a 12-well plate and transfected
with the indicated plasmids. After 24 h, the cells were washed once
with PBS-TX (PBS containing 0.1% Triton X-100), followed by fixing
in ice-cold methanol at rt for 15 min. After washing twice with PBS-TX,
the nuclei were stained with 1 μg/mL DAPI (Sigma) in PBS-TX
at rt in the dark for 5 min. After washing twice with PBS-TX, cover
glasses were mounted onto microscope slides with the cell side down.
The images were acquired on a Zeiss 880 inverted confocal microscope
using a 40× oil lens.

2x10⁴ MCF7 cells were seeded in 24-well glass-bottom SensoPlate™ (662892, Greiner) for 48h. 1h prior to imaging, media was removed and replaced with complete DMEM containing 50nM LysoTracker™ Red DND-99 (L7528, Invitrogen™) and 100nM MitoTracker™ Green FM (M7514, Invitrogen™). Then, media was removed and replaced with fresh media. Living cells were imaged in a Zeiss 880 Inverted confocal microscope (Zeiss) using a ZEN fluorescence microscopy system (Zeiss) and a 63x Plan-Apochromat objective. Laser properties, acquisition mode and detectors were manually adjusted for each experiment.

For fixed samples, all images were captured using an Olympus FV1000 Laser Scanning Microscope with Becker and Hickel FLIM system and Olympus Fluoview FV1200 using FV10-ASW software (version 4.2). Foci counting was performed using FIJI (Image J version 2.0.0-rc-65/1.52a or 2.1.0) and the GDSC (FindFoci) ImageJ plugin^{60 (link)}. Only the foci that overlap with the DAPI staining were analysed. Live-cell images were captured using a Zeiss 880 inverted confocal microscope with Zen Black software (version 14.0.22.201). Images were captured every 4.5 min (Fig. 5b) or 5 min (Fig. 5d) for at least 6 h.