Anti human igm fitc

Mononuclear cells from healthy donors and SS patients were enriched for B cells by magnetic separation with 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) prior to purification. Single CD19⁺CD21^lowCD10⁺IgM^hiCD27⁻ new emigrant/transitional, CD19⁺CD21⁺CD10⁻IgM⁺CD27⁻ mature naïve and CD19^hiCD21^−/lowCD10⁻CD27⁻ referred to henceforth as CD21^−/low B cells were sorted on a FACSAria (BD Biosciences) into 96-well PCR plates and immediately frozen on dry ice.

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).