Spheroid coculture and imaging
For coculture with PBMCs, PBMCs (HLA-matched if possible) pre-stimulated
with IL-2 were added 3 days after spheroid formation at the ratio of
10:1. On the day of coculture, PBMCs were stained with MERCK Red Stain
PKH26 following manufacturer instructions, and spheroids were stained
with 0.1 uM Invitrogen Cell Event, followed by drug administration.
Image acquisition was performed once a day for 3 days. For the blocking
assay, PKH26-stained and CellEvent-stained spheroids were cocultured in
the presence or absence of anti-CXCL10 neutralizing antibody (10 µg/mL;
RandD systems; catalog MAB266). Z-stack analysis was done by
transferring the individual spheroid to a flat-bottom clear-bottom
96-well plate which helped facilitate imaging. Images were obtained at
10 µm increments on the GE InCell6000 instrument using a 10x objective
with an aperture of 0.6-0.8 AU. Then using free software provided by
ImageJ (made available by the National Institutes of Health [NIH]),
stacking of the 10 images was performed, followed by application of
maximum intensity projection along the Z axis by the Fiji plug-in
“Particle Analyzer”. 3D pictures were generated using “Clear
Volume”.