Ethidium homodimer

Cells were plated in low-binding 96-well plates at 150 cells/well and maintained in regular growth media for 7 days. Vybrant Violet Dye (0.5 µM, ThermoFisher Scientific) was added to the cell and incubated at 37 °C for a minimum of 30 min, and Ethidium Homodimer (0.3 µM, Biotium) was then added before analysis. The MetaXpress High Content Image Acquisition platform (Molecular Devices) was used to capture images of a total of eight wells for each cell line, and only the images of the entire cell mass was used for image analysis. ImageXpress image analysis software (Molecular Devices) was used to identify and count the cells stained with either Vybrant Violet (Cy3, blue) or Ethidium Homodimer (Cy5, red). Data was reported as a log2 ratio of live to dead cells (Cy3:Cy5).

DLBCL were incubated with 10 µM CMAC CellTracker (ThermoFisher) in serum-free media for 45 min and washed twice with media prior to pelleting and resuspending in hydrogel. NK cells were incubated with 2 µM CFSE CellTracker (Thermofisher) and resuspended with DLBCL for co-culture conditions. NK cells were loaded at a 1:2 ratio to DLBCL. NK cells were loaded at 5 to 7.5 million cells/mL, while DLBCL was loaded at 10 to 15 million cells/mL. For treated conditions, Rituximab was added to perfused media at 1 µM and CHOP was added at 250 ng/mL. Prior to viability imaging, devices were perfused with 8 µM ethidium homodimer (Biotium, Freemont, CA) for ~3 h. Spheroids were differentiated between low density and high density, with high density having >5 cells/mm².
For immunofluorescent imaging, cells were labeled with fluorescently conjugated antibodies in droplets. For DLBCL, the antibodies used were anti-CD47 conjugated to PerCP-Cy5.5, anti-PDL1 conjugated to Alexa Fluor 594, and anti-CD20 conjugated to Brilliant Violet 421 (Biolegend). For NK cells, anti-SIRPα conjugated to FITC, anti-PD1 conjugated to Alexa Fluor 647, and anti-CD16 conjugated to PE were used for labeling (Biolegend). All images were taken at ×20 magnification.

Cell viability was assessed both with the trypan blue exclusion test and Live/Dead flow cytometry. For the flow cytometry analysis, MSCs in each media formulation were harvested at P5 via digestion with 0.05% trypsin and transferred into a 50-ml conical tube for centrifugation at 200 g for 4 min at room temperature. Following aspiration of excess media, cells were either washed three times with phosphate-buffered saline (PBS) with calcium and magnesium(+/+) and PBS without calcium and magnesium (−/−) or once with PBS (−/−) followed each time by a centrifugation cycle. MSCs were counted using an automated cell counter and stained with 0.4% Trypan blue solution (VWR, Radnor, PA). One million MSCs cultured in FBS or ePL culture media were resuspended in 1 ml PBS and stained with 4 μM ethidium homodimer (Biotium, Fremont, CA) and 2 μM Calcein Blue AM (Thermo Fisher Scientific, Waltman, MA). MSCs stained with either ethidium homodimer or Calcein Blue AM alone were used as control groups. As a negative control, MSCs were harvested, fixed with 4% paraformaldehyde (PFA) for 20 min on ice, washed with PBS, and stained with both ethidium homodimer and Calcein Blue AM. Samples were analyzed by flow cytometry and 50,000 events were collected per sample. Data were analyzed by Flow Jo software (NIH).