Pe cy7 anti cd45ra clone hi100

Fresh peripheral blood samples from de-identified RRMS patients were collected under the approvement by the Northwestern University Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using ISOLYMPH (Cat #759050, CTL). The PBMCs were stained with Aqua LIVE/DEAD fixable staining reagents (Cat #L34957, Life Technologies), human FcR blocking reagent (Cat # 130-059-901, Miltenyi Biotec), APC anti-CD3 (clone OKT3, BioLegend), Percp-cy5.5 anti-CD4 (clone OKT4, BioLegend) PE-Cy7 anti-CD45RA (clone HI100, BioLegend), BV650 anti-CD45RO (clone UCHL1, BD Biosciences), Pacific Blue anti-CXCR3 (clone G025H7, BioLegend) and BB515 anti-CCR6 (clone 11A9, BD Biosciences). Then CD3⁺CD4⁺CD45RA⁻CD45RO⁺CCR6⁻CXCR3⁺ (Th1), CD3⁺CD4⁺CD45RA⁻CD45RO⁺CCR6⁺CXCR3⁺ (Th1/Th17) and CD3⁺CD4⁺ CD45RA⁻CD45RO⁺CCR6⁺CXCR3⁻ (Th17) cells were sorted. After sorting, the cells were amplified by stimulated with anti-CD3/CD28 Dynabeads for 7 days.