Flow Cytometric Analysis of IDO-1 Expression in hPDLSCs

The 2.5 × 10⁵ hPDLSCs were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (ebioscience, Waltham, USA) and were subsequently stained with PE-conjugated mouse anti-human IDO-1 antibody (clone eyedio, ebioscience, Waltham, USA). Cells stained with PE-conjugated mouse IgG1 K immunoglobulin isotype control (ebioscience, Waltham, USA) served as reference. After staining, hPDLSCs were resuspended in 200 µl FACS buffer (3% bovine serum albumin and 0.09% sodium azid in 1xPBS) and analyzed by flow cytometry using the FACSCalibur Flow Cytometer. Fluorescence was excited by an argon laser at 488 nm. In total, 10,000 cells were counted per group. The percentage of IDO-1 positive cells and the corresponding mean fluorescence intensity (m.f.i.) were determined using CellQuest 3.3. software as described previously [24 (link)]. Representative dot plots and one-parameter histograms that demonstrate the gating/analysis strategy are provided in the “Supplementary Materials” (Figure S3).