Anti cd127 percp cy5

The following antibodies were used for flow cytometric stainings anti-TACI PE (clone 1A1), anti-CD19 APC-Cy7, anti-CD27 PerCP-Cy5.5 or APC, anti-CD10 PE-Cy7, anti-IgM FITC, anti-CD21 APC, anti-CD69 PE-Cy7, anti-CD86 PE, anti-CD4 APC-Cy7, anti-CD25 PECy7, anti-CD127 PerCP-Cy5.5, anti-CD45RO Pacific Blue, anti-CXCR5 PerCP-Cy5.5, anti-PD-1 PE-Cy7, anti-CD25 PE, anti-CD25 PE-Cy7 (all from BioLegend, San Diego, Calif), anti-CD3 eFluor 605NC, anti-CD21 BD Horizon V450 (Becton Dickinson) and goat polyclonal anti-TACI biotin (R&D Systems). Intracellular staining with anti-Foxp3 Alexa Fluor 488 and anti-BCL6 PE (eBioscience, San Diego, Calif) was performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), in accordance with the manufacturer's instructions.

Mononuclear cells from healthy donors and patients with SSc were enriched for B cells by magnetic separation with anti-CD20 microbeads (Miltenyi Biotech) and stained with anti-human CD19-Pacific Blue, anti-human CD27-PerCP Cy5.5, anti-human CD10-PE-Cy7, anti-human CD21-APC, anti-human IgM-FITC (Biolegend). Single CD19⁺CD21^lowCD10⁺IgM^hiCD27⁻ new emigrant/transitional and CD19⁺CD21⁺CD10⁻IgM⁺CD27⁻ mature naïve B cells were sorted on a FACSAria (BD Biosciences) into 96-well PCR plates and immediately frozen on dry ice.
The following antibodies were used for T cell phenotyping: anti-CD4 APC-Cy7, anti-CD25 PE-Cy7, anti-CD127 PerCP-Cy5.5 (all from Biolegend), and anti-CD3 eFluor 605NC (eBioscience). Intracellular staining with anti-FOXP3 Alexa Fluor 488 (clone PCH101; eBioscience) was performed using the FOXP3/Transcription Factor Staining Buffer Set in accordance with the manufacturer’s instructions (eBioscience).

Flow cytometry sample acquisition was performed on a LSR Fortessa (BD Biosciences, Mountain View, Calif). The following antibodies were used for flow cytometric stainings: anti-TACI PE (clone 1A1), anti-CD19 APC-Cy7, anti-CD27 AF700, anti-CD4 APC-Cy7, anti-CD8 BV711, anti-CD25 Pe/Dazzle 594, anti-CD127 PerCPCy5.5 (all from BioLegend, San Diego, Calif), anti-IgM PerCPCy5.5 and anti-CD3 eFluor 605NC (BD). Intracellular staining for FOXP3 Alexa Fluor 488 (clone 150D; Biolegend) was performed using the FOXP3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, Calif) in accordance with the manufacturer’s instructions. Subset analysis was performed with FlowJo software (Tree Star, Ashland, Ore).