Cd19 fitc

Peripheral blood mononuclear cells (PBMCs) were obtained from blood. After Ficoll-Isopaque density centrifugation (Rafer, Zaragoza, Spain), collected cells were washed twice with ice-cold PBS, followed by centrifugation at 2000 rpm for 5 min. Next, cells were labeled with antibodies to CD19 – FITC (Miltenyi Biotec, clone LT19), CD27 – APC (Miltenyi Biotec, clone M-T271), IgD – PE (SouthernBiotech, Cat. No. 2032-09) and IgM – PerCP/Cy5.5 (BioLegend, clone MHM-88) for 20 min on ice in a staining buffer (PBS with 4% FBS and 2 mM EDTA). Naïve B cells (CD19⁺ CD27^– IgD⁺) and unswitched memory B cells (CD19⁺ CD27⁺ IgD⁺) were obtained by FACS sorting on a MoFlo Astrios (Beckman Coulter). Purified samples were pelleted and stored at −80°C.
For isolation of naïve autoreactive B cells. Total B cells were isolated from PBMCs using positive selection with MACS CD19 microbeads (Miltenyi Biotec). Next, cells were stained with CD27-APC (Miltenyi Biotec, clone M-T271), IgD – PE (SouthernBiotech, Cat. No. 2032-09), HLA-DR – PE-Cy7 (eBioscience, clone LN3), 9g4 primary ab (igm Bioscience) and donkey anti-rat IgG (H + L) – Alexa Fluor 488 (invitrogen). 9g4+ naïve B cells (CD27^– IgD⁺ 9g4⁺) and 9g4- naïve B cells (CD27^– IgD⁺ 9g4^–) were obtained by FACS sorting on a BD FACSAria II (BD Biosciences). Purified samples were pelleted and stored at −80°C.

CD146⁺/CD45^-/HLA-DR^--MSC were isolated from noncultured BM-MNC using multicolor FACS using standard protocols (ARIA II; BD Biosciences). Immunophenotype of CFU-F–derived MSC and CD146⁺/CD45^-/HLA-DR^--MSC was analyzed using flow cytometry (LSRFortessa, FACSDiva software; BD Biosciences) according to a standardized protocol. The evaluated antigens were selected according to the International Society for Cellular Therapy consensus.⁶ (link) For FACS and flow cytometric analyses the following mouse anti-human antibodies were used: CD90-PE, CD105-FITC, CD146-PE, CD45-FITC, HLA-DR-FITC, CD34-FITC, CD73-PE, CD14-PE, and CD19-FITC (all Miltenyi Biotec).