Cd19 bb700

All experiments to establish a median mismatch index (MMI) were done using a human CD4 evaluation kit (BD Biosciences, 566352) except for the Nova Yellow 690, which was purchased from Thermo Fisher Scientific (H001T03Y05). The following fluorochromes were used: BUV615, BUV661, and BUV737 (under 355-nm excitation); BV421, BV650, and BV711 (under 405-nm excitation); FITC and BB700 (under 488-nm excitation); PE-CF594, PE-Cy5, and Nova Yellow 690 (under 561-nm excitation); and allophycocyanin and Alexa Fluor 700 (under 640-nm excitation). DAPI, CD14 PE-Cy5 (BioLegend, 301863), CD3 allophycocyanin-R700 (BD Biosciences, 565120), CD4-allophycocyanin (BD Biosciences, 566915), CD8-BV786 (BioLegend, 344740), CD19 BB700 (BD Biosciences, 566397), and CD45RA-BV711 (BioLegend, 304137) were used to generate a small panel for testing the effect of compensation beads on biological interpretation in terms of the position of these populations on the plot.

Bone marrow of Myce and littermate wild‐type control were harvested into single‐cell suspension with red cell lysis and stained with CD19‐Pacific Blue (1D3, BD, NJ, USA), B220‐PE cy7 (RA3‐6B2, BD), CD24‐APC (M1/69, in house), IgM‐FITC (331.12, in house), CD43‐PE (S7, BD). Spleen red cell lysed single cell suspensions were stained with CD19‐BB700 (1D3, BD), B220‐APC (Thermo Fisher, Waltham, USA), CD23‐PE cy7 (B3B4, Thermo Fisher), CD21‐BV421 (7E9, BioLegend, San Diego, USA), CD138‐PE (281–2, BD). The peritoneal cavity wash was stained with CD19‐PE (ID2, in house), B220‐APC (RA3‐6B2, Thermo Fisher), CD23‐PE cy7 (B3B4, Thermo Fisher), CD5‐BB700 (53–7.3, BD), CD11b‐eFlour450 (M1/70, Thermo Fisher). Lymph node and thymus of single cell suspensions were stained with CD19‐BUV395 (1D3, BD), (TCRβ‐PE (H57‐597, BD), CD4‐BV421 (GK1.5, BioLegend) and CD8a‐PerCp/Cy5.5 (53–6.7, eBioscience, San Diego, USA). Flow cytometry analysis was performed on a BD LSRFortessa X‐20 analyzer (BD).

PBMC were plated and stimulated with CpG-ODN 2006 (Aurogene Srl) alone or in the presence of 100 μl of CM-hAMSC as described above. After 5 days of culture, cells were collected and protein expression of the transcription factors BCL6, PAX-5, IRF-4, and BLIMP-1 was analyzed by flow cytometry. After exclusion of dead cells by eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific) staining, cells were stained for 20 min at 4°C with CD19 BB700 (SJ25C1, 1:200), CD3 BV510 (UCHT1, 1:100), CD14 BV510 (MΦP9, 1:200), CD27 PE-Cy7 (M-T271, 1:100), all from BD Biosciences. After washing in stain buffer, the cells were fixed and permeabilized with Transcription Factor Buffer Set (BD Biosciences) for 40 min at 4°C, according to the manufacturer's instructions. Then, cells were stained for 40 min at 4°C with specific antibody against BCL6 BV421 (K112-91, 1:30), PAX-5 PE (1H9, 1:100), IRF-4 PE (Q9–343, 1:100), BLIMP-1 Alexa Fluor® 647 (6D3, 1:100). Finally, the cells were washed with Transcription Factor Buffer Set (BD Biosciences), acquired on a FACSAria III (BD Biosciences), and analyzed using FCS express v5.0 (DeNovo Software, Los Angeles, CA, USA).