Environmental chamber

Multi-parametric live cell confocal imaging was operated by an inverted motorized microscope (Nikon TiE, Japan) enclosed by an environmental chamber (OkoLab, Italy). Laser illumination was controlled by iLas2 (GATACA Systems, France) based on Metamorph (Molecular Devices, USA). The measurement and analysis of nanoparticle uptake (vanishing time) on PANC-1 apical cortex and their accumulated numbers are explained in Supplementary Figure 9,10.

U2-OS cells were grown and transfected as described above with plasmids encoding an N-terminal fusion of H2B to either mEGFP [18 (link)], AausFP1, or mAvicFP1, all with identical linker sequences. Prior to imaging, cells were stained with SiR-Hoechst (Cytoskeleton) following the manufacturer’s instructions and maintaining a low concentration of stain in the medium throughout imaging. Cells were imaged on a Leica TCS SP8 system with an Okolab environmental chamber, as described above, starting 12–24 hours post-transfection. Images were collected every 2 minutes for >72 hours using 488-nm excitation with green emission to detect the H2B fusions, and with 633-nm excitation and far-red emission for the SiR-Hoechst stain to detect all DNA. For analysis, cells were selected from those expressing H2B and that underwent 1 cell division in the first half of the experiment. Control cells were selected from those neighboring the selected H2B-FP-expressing cells. The interval between cell divisions, defined as the time between visible chromosome separation, was recorded for the 2 daughter cells of each original cell.

Experiments were carried out as detailed previously.^{48 (link)} Imaging was performed with a × 40 ELWD Plan Fluor objective (NA: 0.6, Nikon, Melville, NY, USA) on a Nikon Ti-E perfect focus inverted microscope equipped with a spinning disk confocal CSU-X1 (Andor, Oxford Instruments, Belfast, UK), motorized X,Y stage (Nikon), environmental chamber (OkoLab, Pozzuoli, Italy), and iXon3 897 EMCCD camera (Andor, Oxford Instruments), controlled by the NIS-Elements software (Nikon). All analysis was performed using MATLAB (version R2012b, Mathworks, Natick, MA, USA) on 16-bit grayscale images of eGFP-BCL-2 family proteins or mCherry-BH3-only proteins. The mitochondrial compartment was identified as eGFP-localized, small, isolated structures (>~4 μm² and <80 μm²) above local background intensity. In contrast to Wong et al.,^{48 (link)} nuclear-specific areas were not observed. Therefore, the entire cell was identified with a single intensity threshold applied to the eGFP fluorescence, excluding areas identified as mitochondria. Average intensities were reported on an image-wide basis, normalized to the total area of each compartment identified. Ratio of mCherry mitochondrial intensity/cytoplasmic intensity was calculated, and data were normalized and plotted as described.^{48 (link)} Data were analyzed from 10 fields of view per condition in two separate experiments.

Spheroids were imaged every 4 hours for 5 days at 37°C and 5% CO₂. Z-stacks were acquired in phase, 488 and 647 channels at 10 μm steps (140 μm total) with a 10× Plan Fluor objective (NA: 0.3, Nikon). Imaging was performed on a Nikon Ti-E perfect focus inverted microscope equipped with a spinning disk confocal CSU-X1 (Andor, Oxford Instruments) motorized X, Y stage (Nikon), environmental chamber (OkoLab) and Prime 95B sCMOS camera (Teledyne Photometrics), all controlled by NIS-Elements software (Nikon). Z-stacks were processed into maximum intensity projections, spheroids were traced in the phase image and area was measured at every time point, then normalized to the initial measurement at t=0. Total integrated intensity of the CellTrace Far Red fluorescence was measured within spheroid ROIs in the 647 channel and normalized to t=0 measurements. 6 donors with 3 wells each were assayed with or without XmAb24306 treated NK cells, or spheroids alone.

Cells were washed and incubated in phenol red-free medium and imaged on a Nikon TiE inverted microscope with an Okolab environmental chamber at 37 °C, 5% CO₂. Images were obtained and processed using NIS Elements (Nikon).