*4.2. Lymph Node Collection and Processing from Kidney Transplantation Recipients*

LNs were collected from surgical residual material of kidney transplantation recipients (*n* = 10) during implantation of the kidney. Before anastomosing the arteria and vena renalis, the iliac artery and vein were dissected free. The resulting residual tissue that was removed in this procedure often contains LNs. Though all patients were treated with a quadruple immunosuppressive therapy, only the first dose of CD25mAb was administered before the procedure and we have demonstrated that CD25mAb (basiliximab; Novartis Pharma, Amsterdam, The Netherlands) was not detectable in LN cells and that ex vivo CD25 expression on cells could be blocked with CD25mAb [62]. LNs were carefully cleaned of fat and connective tissue, then cut into small pieces (< 0.5 cm) after which a cell suspension was obtained by grinding the material through a flow-through chamber. The remaining tissue stroma was used in this study and digested as described before [45] to isolate and analyze LNSCs directly ex vivo. In summary, LN stroma was digested using the enzymatic mixture of 0.2 mg/mL collagenase *p* (Roche, Woerden, The Netherlands), 0.8 mg/mL Dispase II (Roche, Woerden, The Netherlands), and 0.1 mg/mL DNAse I (Roche, Woerden, The Netherlands) in RPMI medium (Invitrogen, Landsmeer, The Netherlands) without serum. Cell suspensions were filtered through a 70-μm nylon cell strainer and directly stained for flow cytometry.
