M500kcaf0y

Peripheral blood mononuclear cells were isolated from the blood of healthy volunteers from the Etablissement Français du Sang (Lyon, France) or from the Belgian Red Cross Flanders by Ficoll gradient centrifugation (Eurobio, Courtaboeuf, France). Cell sorting methods are detailed in Supplementary Appendix. Purified NK cells and non-classical monocytes were then cocultured with glomerular endothelial cells. In a first experiment, NK cells were cocultured with the human conditionally immortalized glomerular endothelial cell line (38 (link)) (ciGENC; HLA-A2), in combination with either anti-HLA-A2 DSA-containing serum or control human serum. After 4 h of coculture, supernatants were collected. In a second experiment, NK cells and non-classical monocytes were cocultured with primary glomerular endothelial cells (GENC, Cell Systems, USA), after incubation with either anti-HLA-A, -B, -C purified antibody (BD Biosciences, San Jose, CA, USA; cat #560187), or control isotype (BD Biosciences, cat #553447). After 24 h of coculture, supernatants were collected. Details on the two experiments are specified in the Supplementary Appendix.
Levels of 27 cytokines, chemokines, and growth factors in the supernatants were assessed using the same 27-multiplex analysis as used for the clinical samples, following the manufacturer’s instructions (Bio-Rad, M50-0KCAF0Y; Bio-Rad, Nazareth, Belgium).

Concentrations of SIM and epithelial and endothelial factors were determined using Luminex-based multiplex arrays, i.e., human 27-Plex (M500KCAF0Y, BioRad, Hercules California, USA), and Cancer Panel 2 (171AC600M, BioRad) in a miniaturized variant of the manufacturer's instructions. As little as 1–2 μl of perilymph fluid were diluted with sample diluent (1:20) and incubated with multiplex beads for 45 min followed by two washings steps, cocktail of biotinylated secondary mAbs for 30 min and after final washing steps, streptavidin-PE was added. Greater than fifty beads per sample per analyte were detected using the BioPlex Manager 6.2 Software and concentrations were calculated according to individual standard curves for each analyte ranging from ~20 ng/ml to the detection limit of ~2 pg/ml.