Fibroblasts were labeled using a PKH26 red fluorescent cell membrane labeling kit (Sigma,
St. Louis, MO, USA) according to manufacturer’s instructions and injected IP into mice.
Mice were euthanized at six time points at 7-day intervals (i.e. week 1 to week 6). Cells
were retrieved from peritoneal cavity (by lavage), lymph nodes, spleen, lung, and skin.
Single cell suspensions form tissues were prepared using collagenase D (Roche Diagnostics)
digestion (1 mg/ mL, for 30 min at 37 °C) and examined using flow cytometry. To further
characterize the cells that were extracted from lymph nodes, these cells were stained for
CD90.2 (eBioscience) and examined using flow cytometry. Presence of migratory fibroblasts
in lymph nodes was examined using confocal microscopy. To do so, PKH26-labeled
IDO-expressing fibroblasts (2 × 107 cells/mouse) were injected IP into C3 H
mice. After 2 weeks, mice were euthanized and mesenteric lymph nodes were harvested,
freshly frozen and embedded in Cryomatrix (Thermo Scientific, Kalamazoo, MI, USA). Frozen
sections (5 μm thick) from lymph nodes were permeabilized with 0.1% Triton-X-100 in
phosphate buffered saline (PBS), stained with 4’,6-diamidino-2-phenylindole (DAPI) and
visualized using a confocal fluorescence microscope (Axio Observer Z1 inverted confocal
with spinning disk, Carl Zeiss, Jena, Germany). Images were analyzed using Zen software
(Carl Zeiss).
St. Louis, MO, USA) according to manufacturer’s instructions and injected IP into mice.
Mice were euthanized at six time points at 7-day intervals (i.e. week 1 to week 6). Cells
were retrieved from peritoneal cavity (by lavage), lymph nodes, spleen, lung, and skin.
Single cell suspensions form tissues were prepared using collagenase D (Roche Diagnostics)
digestion (1 mg/ mL, for 30 min at 37 °C) and examined using flow cytometry. To further
characterize the cells that were extracted from lymph nodes, these cells were stained for
CD90.2 (eBioscience) and examined using flow cytometry. Presence of migratory fibroblasts
in lymph nodes was examined using confocal microscopy. To do so, PKH26-labeled
IDO-expressing fibroblasts (2 × 107 cells/mouse) were injected IP into C3 H
mice. After 2 weeks, mice were euthanized and mesenteric lymph nodes were harvested,
freshly frozen and embedded in Cryomatrix (Thermo Scientific, Kalamazoo, MI, USA). Frozen
sections (5 μm thick) from lymph nodes were permeabilized with 0.1% Triton-X-100 in
phosphate buffered saline (PBS), stained with 4’,6-diamidino-2-phenylindole (DAPI) and
visualized using a confocal fluorescence microscope (Axio Observer Z1 inverted confocal
with spinning disk, Carl Zeiss, Jena, Germany). Images were analyzed using Zen software
(Carl Zeiss).