Gallios 10 color flow cytometer

Mononuclear cells (peripheral blood mononuclear cells [PBMCs]) were isolated using Pancoll Human (PAN Biotech, Darmstadt, Germany). Single-cell suspensions of renal allograft specimens were obtained by combined enzymatic (Collagenase-IV and DNAse, Applichem, Darmstadt, Germany) and mechanical (GentleMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) dissociation. Circulating lymphocytic subsets were analyzed on a Beckman Coulter Gallios 10-color flow cytometer using a panel of fluorescent-labeled antibodies (Table S3). The remaining PBMCs were frozen and stored in liquid nitrogen until needed for further analysis. For intracellular staining of IL-10 and GrB, frozen samples were thawed and cultured in AIM V Medium (Life Technologies, Darmstadt, Germany) on a 96-well plate. Cells were stimulated with 5 μg/mL Class B CPG ODN 2006 (Miltenyi Biotech) for 18 h. Fifty ng/mL phorbol myristate acetate (PMA) and 750 ng/mL ionomycin were added for the last 5 h, and Brefeldin A (Biolegend) and Monensin (Biolegend, Fell, Germany) were added for the last 2 h. Cell surface staining was performed first. Phycoerythrin (PE)-conjugated IL-10 (Biolegend, Fell, Germany) and AF647-conjugated GrB (Biolegend) were used for intracellular cytokine staining according to the manufacturer's instructions.

PBMC were thawed and immediately centrifuged with PBS to dispose DMSO. TIL were stained directly. The cells were incubated separately for the different stainings and labeled with mAbs at room temperature for 30 min in the dark. After the incubation time, PBMC were washed and collected in 250 µl PBS-containing 0.5% BSA for flow cytometry analysis. All flow cytometry measurements were performed using a Gallios 10-color-flow-cytometer equipped with Kaluza flow cytometry software (both Beckman Coulter). The acquisition and analysis gates were restricted to the lymphocyte gate based on characteristic properties of the cells in forward and side scatter (FSC and SSC). At least 10 5 cells were acquired for analysis.