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2 protocols using cd3 efluor 605nc

1

Comprehensive B and T Cell Phenotyping

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B cells were purified from the patient’s peripheral blood by positive selection using CD20 magnetic beads (Miltenyi Biotec, Cambridge, MA). CD4+ T cells were isolated from the peripheral blood of research subjects using the EasySep human CD4+ T cell enrichment kit (STEMCELL Technologies, Cambridge, MA). The following antibodies were used for flow cytometric staining: CD19 APC-Cy7, CD27 PerCP-Cy5.5, CD10 PE-Cy7, CD21 V450, CD69 PE, CD86 APC, FAS Alexa 647, CD4 APC-Cy7, CD127 PerCP-Cy5.5, CD45RO Alexa 700, CXCR5 PerCP-Cy5.5, PD-1 PE-Cy7, ICOS APC (all from Biolegend, San Diego, CA), CD3 eFluor 605NC (from eBioscience, San Diego, CA), and CD21 BD Horizon V450 (BD). Intracellular staining for FOXP3 Alexa 488 (eBioscience) and T-bet PE was performed using the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) in accordance with the manufacturer’s directions.
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2

Isolation and Characterization of Synovial Tissue Cells from RA Patients

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Cells were isolated from ST biopsies, obtained from 4 late-stage RA patients as described [6 (link)]. Cells were resuspended in RPMI-1640 containing 10% fetal bovine serum (FBS; Lonza, Breda, the Netherlands), 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until analysis. ST cells from all 4 subjects were thawed at the same time and were stained with Fixable Viability Stain 450 (BD Biosciences) according to the manufacturer’s instructions. Following washing with PBS containing 0.5% BSA and 2 mM EDTA, cells were resuspended in the washing buffer with Fc receptors blocking reagent (Miltenyi Biotech) and stained with the following mouse monoclonal anti-human antibodies: LLT1 PE (clone 402659, Cat No FAB3480P, R&D Systems), CD3 eFluor 605NC (Cat No 93–0037), CD19 PE-Cyanine7 (Cat No 25–0199, both from eBioscience), CD161 APC (Cat No 130-092-678, Miltenyi Biotech) for 30 min. at RT. After fixation and permeabilization with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), cells were stained with mouse monoclonal anti-human CD68 FITC antibody (Cat No 11–0689, eBioscience) for 30 min. at RT, followed by washing with the permeabilization buffer (eBioscience). ST cells were analyzed using LSR II flow cytometer (BD Biosciences), and data analysis was performed with Kaluza analysis software (Beckman Coulter).
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