Pe conjugated mouse igg1 k immunoglobulin isotype control

1.25 × 10⁵ harvested hPDLSCs were resuspended in 50 µl FACS buffer and stained with the following antibodies for 20 minutes: PE-conjugated mouse anti-human CD274 antibody (clone B7-H1, ebioscience, Waltham, USA) or PE-conjugated mouse anti-human CD273 antibody (clone B7-DC, ebioscience, Waltham, USA). Unlabeled cells stained with (PE)-conjugated mouse IgG1 K immunoglobulin isotype control (ebioscience, Waltham, USA) served as control. After staining, cells were resuspended in 200 µl FACS buffer. Flow cytometry analysis was performed as described above. The percentage of PD-L1 or PD-L2 positive cells was determined using CellQuest 3.3 software. Representative dot plots that outline the used gating/analysis strategy are shown in the “Supplementary Materials” (Figure S4).

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).

After fixing and permeabilization using the Intracellular Fixation and Permeabilization Buffer Set (eBioscience, Waltham, USA), 2.5x10⁵ hPDL-MSCs were intracellularly stained by 0.06µg of the R-phycoerythrin (R-PE)-conjugated mouse anti-human IDO-1 monoclonal (clone eyedio) antibody (Thermo Fisher Scientific, Waltham, USA) for 30 minutes. Additionally, hPDL-MSCs were labeled with PE-conjugated mouse IgG1 κ immunoglobulin isotype control (Thermo Fisher Scientific, Waltham, USA) which served as control. Subsequently, cells were washed several times and resuspended in 0.5ml 3% bovine serum albumin (BSA, Capricorn Scientific, Ebsdorfergrund, Germany, in 1xPBS + 0.09% sodium azide, Merck Darmstadt, Germany) for acquisition. Flow cytometry analysis was performed using FACSCalibur Flow Cytometer (BD Biosciences, Franklin Lakes, USA) exciting R-PE by an argon laser at 488nm. In total, 10,000 cells were acquired per sample. Gates were set using unlabeled hPDL-MSCs. The gating strategy is described in the Supplementary Material of our previous study (2 (link)). The percentage of IDO-1⁺ hPDL-MSCs and the corresponding mean fluorescence intensity (M.F.I.) were calculated by CellQuest 3.3. software (BD Bioscience, Franklin Lakes, USA).