Cell preparation - Isolation of CD3+/CD3- immune cells
Fresh blood samples (30 mL) were drawn into EDTA tubes (Vacutainer, Becton Dickinson, UK) from patients, HC and ADPKD. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare, Sweden). Subsequently, CD3+ cells were isolated by positive selection using a magnetic cell-sorting system (Miltenyi Biotec GmbH, Germany) according to the manufacturer’s instruction. Following isolation, cells were divided into CD3+ cells (eluted from the columns) and CD3- cells (unlabeled cells). Cells were stained with Live/Dead Fixable Near-IR Dead cell stain kit (ThermoFisher Scientific Inc., USA) for 10 min for analysis of the live cell fraction. Subsequently, the CD3- cells were subjected to two panels stained for B cell- and monocyte surface markers.
The CD3+ PBMCs were subjected to surface marker staining for T cell subsets: CD4+/CD8+ CCR7+ CD45RA+ naïve T cell, CD4+/CD8+ CCR7+ CD45RA- central memory T cell, CD4+/CD8+ CCR7- CD45RA+ effector T cell, CD4+/CD8+ CCR7- CD45RA- effector memory T cell, CD4+ CXCR3+ CCR6- Th1 cell, CD4+ CXCR3- CCR6- Th2 cell, CD4+ CXCR3- CCR6+ Th17 cell and CD4+ CCR4+ CD25+ CD127low regulatory T cells.
To phenotype B cells, cells were stained with anti-CD19, anti-CD27, anti-CD38 and anti-IgD (Biolegend Inc.) monoclonal antibodies. In the monocyte panel cells were stained with anti-CD14 and anti-CD16 (Biolegend Inc.) monoclonal antibodies. To phenotype T cell subsets, cells were stained with anti-CD4, anti-CD8, anti-CD45RA, anti-CCR7, anti-CXCR3, anti-CCR6, anti-CD25, anti-CD127 and anti-CCR4 (Biolegend Inc., UK) monoclonal antibodies. These surface markers have earlier been suggested to identify different subsets of T-, B cells as well as monocytes in peripheral blood (8).
After 30 min staining, cells were washed and acquired on flow cytometry. Cells were analyzed within the gates on forward and side scatter plots and live cells were examined.
Gating strategies for analysis of B cell and monocyte subpopulations are shown in Fig. 1.