Cd19 b cell isolation kit

Peripheral blood mononuclear cells (PBMCs) were purified from healthy donor buffy coats (Department of Transfusion Medicine, Uppsala University Hospital, Sweden) using Ficoll density-gradient centrifugation. The B cells were isolated from PBMCs by positive selection using CD19+ B-cell isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. The cells were cultured in 1 ml volumes in 24-well plates (Nunc) in macrophage serum-free medium (Life Technologies) at the concentration of 3 × 10⁶ B cells/ml. The cells were stimulated with a phosphorothioate-modified CpG A oligonucleotide ODN2216 (CyberGene) at the concentration of 3 μg/ml and incubated for 5 h at 37 °C with 5 % CO₂. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

Circulating T_FH cells were isolated using a two-step procedure: at first, CD4⁺ T cells were enriched from PBMCs with a CD4⁺ T cell isolation kit; then, CXCR5⁺ T cells were purified using a CD185 (CXCR5)-biotin antibody and anti-biotin microbead (all from Miltenyi Biotec; Bergisch Gladbach, Germany) according to the manufacturer’s instructions. B cells were isolated with a CD19⁺ B cell isolation kit (Miltenyi Biotec). Cell purity was above 80% for cT_FH-like cells and it was higher than 97% for B cells, as determined by flow cytometry (healthy controls, n = 3; patients with SLE, n = 3).

PBMCs were separated by gradient centrifugation with a Lymphoprep (Axis-Shield; Oslo, Norway) according to the manufacturer’s instructions. CD4⁺ T cells and CD19⁺ B cells were enriched from PBMCs using a CD4⁺ T cell isolation kit and CD19⁺ B cell isolation kit, respectively (Miltenyi Biotec; Bergisch Gladbach, Germany) according to the manufacturer’s instructions. For sorting of Tfh cell subsets, enriched CD4⁺ T cells were stained with anti-CCR6-PE (11A9, BD Biosciences), anti-CXCR3-APC (1C6, BD Biosciences), anti-CXCR5-PerCP/Cy5.5 (J252D4, BioLegend), anti-CD45RA-BV421 (HI-100, BioLegend), and anti-CD4-HorizonV500 (RPA-T4, BD Biosciences). Tfh cell subsets were sorted from CD4⁺ T cells as Tfh2 (CXCR3^-CCR6^-), Tfh1 (CXCR3⁺CCR6^-), or Tfh17 (CXCR3^-CCR6⁺) cells among CXCR5⁺CD45RA^-CD4⁺ T cells [19 (link)]. For sorting of naïve B cells, enriched CD19⁺ B cells were stained with anti-IgD-PE (IA6-1, Biolegend) and anti-CD27-APC/Cy7 (O323, BioLegend). Naïve B cells were sorted from CD19⁺ B cells as IgD⁺CD27^-CD19⁺ cells. Tfh cell subsets and naïve B cell sorting was conducted with a FACS Aria III (BD Biosciences) according to the manufacturer’s instructions. Cell purity of naïve B cells determined by flow cytometry was greater than 98 % and cell purity was greater than 93 % for Tfh cell subsets.

Specific cell populations were purified or depleted from hPBMC by using MACS MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany) following the manufacturer’s instructions. PDCs, B-lymphocytes, T CD4 lymphocytes, monocytes and Natural Killer (NK) cells were purified from hPBMC by using the separation magnetic system: BDCA-4 cell isolation kit, CD19 B cell isolation kit, CD4 MicroBeads, CD14 MicroBeads and CD56 MicroBeads respectively (Miltenyi Biotec). PBMC specific cell depletion was performed by positive selection. The purity (85–97%) of isolated cells was evaluated by flow cytometry analysis. All samples, assays and data acquisition were according to MIATA guidelines.