MSC Functional Analysis—Indoleamine 2,3 Dioxygenase (IDO) Activity Assay

MSCs from each cell-line were thawed and cultured for 48 h with a media change at 24 h. MSCs were harvested using TryplE then seeded at a density of 40 000 cells/cm² in a 96 well plate. After 24 h, the medium was replaced in each well with complete medium containing 10 ng/mL IFN-γ (Life Technologies). After an additional 24 h, conditioned medium was collected and frozen at −20 °C, and cells were fixed with 4% PFA. Each medium sample was thawed and 100 μL transferred into a 96 well plate. Trichloroacetic acid was used to precipitate excess protein. A total of 75 μL of the supernatant was collected and transferred to a separate 96 well plate. Ehrlich’s Reagent was then added to each well to detect L-kynurenine levels using a SpectraMax iD5 (molecular devices) plate reader. Levels of L-kynurenine were determined using a standard curve. To normalize L-kynurenine values to cell numbers for each replicate well (n = 5 wells) within an experimental group, we performed automated image analysis to quantify cell nuclei in the wells from which the conditioned medium was collected. Following fixation, MSCs were washed with PBS twice, and stained with Hoechst (10 µg/mL) in 0.1% Tween20 PBS solution for 1 h. Cells were then imaged on a Cytation 5 high-content imaging system (Biotek, Winooski VT) and cell counts were determined using CellProfiler to normalize the amount of L-kynurenine per cell.