Ti e perfect focus inverted microscope

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.

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.