Isotype matched control igg antibodies

Circulating Tang cells were characterized by flow cytometry. Briefly, 300 μl of fresh whole peripheral blood was stained with anti-CD3 allophycocyanin-H7 (APC-H7), anti-CD31 fluorescein isothiocyanate (FITC) and anti-CXCR4 phycoerythrin (PE) antibodies or isotype-matched control IgG antibodies (all from BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Subsequently, red blood cell lysis was performed and cells were analyzed. At least 30,000 CD3⁺ T lymphocytes events were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences). CD3⁺ T lymphocytes double-positive for CD31 and CXCR4 were considered Tang cells. For the evaluation of circulating EPC, CD34⁺ cells were enriched from peripheral blood mononuclear cells from SSc patients and HC using the CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with anti-CD34 FITC, anti-VEGF receptor-2 (VEGFR-2) Peridinin Chlorophyll Protein-Cy5.5 (PerCP-Cy5.5) (both from Miltenyi Biotec) and anti-CD133 PE (BD Biosciences) antibodies. At least 5,000 events in the CD34⁺ enriched population gate were acquired at low rate and EPC were identified as CD34⁺CD133⁺VEGFR-2⁺ cells.

PB mononuclear cells (PBMCs) were isolated from sodium heparinized whole blood by Ficoll-Paque density gradient centrifugation (GE Healthcare, Pittsburgh, PA, USA). Then, the phenotypes of lymphocytes were determined using flow cytometry. Briefly, PBMCs were stained with the following fluorochrome conjugated monoclonal antibodies: anti-CD3-peridin chlorophyll protein (Percp), anti-CD4-Percp, anti-CD8-Percp, anti-CD31-phycoerythrin (PE), anti-CXCR4-allophycocyanin (APC), and isotype-matched control IgG antibodies (all from BD Biosciences, San Diego, CA, USA) for 30 min at room temperature, according to the manufacturer's instructions. After being washed with PBS, a minimum of 20,000 events per tube was acquired using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences) and FlowJo 7.6.1 software (Tree Star).

The phenotypes of lymphocytes in PB and SF were determined using flow cytometry. Briefly, PBMCs and SFMCs were stained with the following fluorochrome conjugated monoclonal antibodies: fluorescein isothiocyanate- (FITC-) conjugated CD3 (SK7), peridinin chlorophyll protein- (PerCP-) conjugated CD4 (SK3), allophycocyanin- (APC-) conjugated CD8 (SK1), phycoerythrin- (PE-) conjugated CD161 (DX12), and isotype-matched control IgG antibodies (all from BD Biosciences, San Diego, CA, USA) for 30 min at room temperature, according to the manufacturer's instructions. Stained cells were analyzed using FACSCalibur flow cytometer (BD Biosciences), and data analysis was performed with Cell Quest software (BD Biosciences).