Bicoll separating solution

The study protocols were approved by the institutional review board of Seoul National University Hospital and Severance Hospital. Peripheral blood of ESRD patients and healthy controls (HCs) was drawn after obtaining written, informed consent. The methods were performed in accordance with the approved guidelines. Peripheral blood mononuclear cells (PBMC) were isolated from blood by density gradient centrifugation (Bicoll separating solution; BIOCHROM, Cambridge, UK). Total monocytes were negatively separated from PBMC with pan-monocyte microbeads (Miltenyi Biotec, Auburn, CA), if no special mention in the figure legend. Total CD4⁺ T cells and CD4⁺ memory T cells were negatively enriched from PBMC using human CD4 T cell enrichment kit and human memory CD4 T cell enrichment kit (STEMCELL Technologies, Vancouver, Canada), respectively. CD4⁺CD28⁺ and CD4⁺CD28⁻ T cells were separated from CD4⁺ memory T cells using a human CD28 microbead kit (Miltenyi Biotec) as described in user’s instructions. CD28⁻ cells were retained in the run-through fraction including CD28 unlabeled cells.

Peripheral blood mononuclear cells (PBMCs) were prepared from human buffy coats (DRK-Blutspendedienst Baden-Württemberg-Hessen, Frankfurt, Germany) using Bicoll Separating Solution (Biochrom). Subsequently, CD14⁺ cells, i.e. monocytes, were isolated by magnetic cell sorting using microbeads for human CD14 (Miltenyi Biotec). For spheroid co-cultures, 5 days old spheroids were cultured with 7.5 x 10⁴ CD14⁺ cells for additional 2 days to allow for infiltration. To further validate the infiltration, CD14⁺ cells were labeled for 10 min with CFSE (eBioscience) prior to addition to the spheroids and evaluated fluorescence microscopically at the end of the infiltration.
For monolayer co-cultures, isolated PBMCs were seeded at a density of 80% in 15 cm dishes in RPMI 1640 medium including 100 U/ml penicillin and 100 μg/ml streptomycin. After adhesion, medium was changed to the same medium including 5% AB-positive human serum (DRK-Blutspendedienst Baden-Württemberg-Hessen, Frankfurt, Germany) to allow for differentiation of the adherent monocytes to MФs. Medium was changed every 2–3 days for 7 days, after which 4 x 10⁶ MCF7 cells were added for co-culture for 48 hours in MCF7 medium. Conditioned media of the MФs, the MΦ-MCF7 co-cultures, or pure MCF7 cells were collected after 48 hours, centrifuged at 1000 x g for 5 minutes at 4°C and stored at -80°C until further use.

Paraformaldehyde was purchased from Affymetrix (Santa Clara, CA). Anti-human TACI antibody (clone 165604) and the respective isotype control were from R&D Systems (Minneapolis, MN). CD41a-PeCy5, CD61-FITC and CD62P-FITC were from BD Pharmingen (San Diego, CA). The goat anti-mouse PE conjugate was from Dako (Glostrup, Denmark). Bicoll Separating Solution was purchased from Biochrom AG (Berlin, Germany). Recombinant human BAFF (rhBAFF) and recombinant human APRIL (rhAPRIL) was from PeproTech (Rocky Hill, NJ, USA). Thrombin Receptor Activator Peptide 6 (TRAP-6), collagen and ADP was purchased from SigmaAldrich (St. Louis, MO). Citrate buffer contained 10 mM sodium citrate, 150 mM NaCl, 1 mM EDTA, 1% dextrose, pH 7.4. GI254023 was from Tocris (Bristol, UK) and Batimastat was from Calbiochem (Darmstadt, Germany).