Proportion of Tfh、Tph and Treg cells in peripheral blood of 53 patients with rheumatoid arthritis and 50 patients with rheumatoid arthritis complicated with Hashimoto's thyroiditis

Tfh and Tph(1) Take 100 μ L of fresh blood from the subject and add it to a 5mL flow tube, then insert it into an ice box for later use.(2) Add FITC anti-human-CD45RA, PE anti-human-CD197 (CCR7), APC anti-human-CD4, APC AlexFluor700 anti-human-CD8, APC AlexFluor750 anti-human-CD3, KromeOrange anti-human-CD45, PerCP-Cyanine5.5 anti-human-CD279 (PD-1) from BECKMAN COUPTER, and PE/Dazzle™ 594 anti-human-CD185(CXCR5) from Biolegend, 10 μ L each.(3) After shaking and mixing, incubate at 4 ℃ in the dark for 15 minutes.(4) After incubation, add 500 μ L of red blood cell lysis buffer.(5) After shaking and mixing, let it stand at 4 ℃ in the dark for 15 minutes.(6) Wash the cells with 500 μ L PBS, shake and mix well, then centrifuge at room temperature for 5 minutes at 300g, and discard the supernatant.(7) Add 2mL of PBS to wash the cells, shake and mix well, then centrifuge at room temperature for 5 minutes at 300g, and discard the supernatant.(8) Add 300 μ L PBS, shake and mix well, and and detect by flow cytometry(BECKMAN COULTERDxFLEX).(9) Analyze the collected data using FlowJo-v10.8.1 softwareTreg(1) Take 100 μ L of fresh blood from the subject and add it to a 5mL flow tube, then insert it into an ice box for later use.(2) Add 10 μ L of FITC anti-human-CD45RA, PE anti-human-CD25, PE-Cyanine7 anti-human-CD127, APC anti-human-CD4, APC-A750 anti-human-CD3, and PacificBlue anti-human-HLA-DR from BECKMAN COUPTER in sequence.(3) After shaking and mixing, incubate at 4 ℃ in the dark for 15 minutes.(4) After incubation, add 500 μ L of red blood cell lysis buffer.(5) After shaking and mixing, let it stand at 4 ℃ in the dark for 15 minutes.(6) Wash the cells with 500 μ L PBS, shake and mix well, then centrifuge at room temperature for 5 minutes at 300g, and discard the supernatant.(7) Add 2mL of PBS to wash the cells, shake and mix well, then centrifuge at room temperature for 5 minutes at 300g, and discard the supernatant.(8) Add 300 μ L PBS, shake and mix well, and and detect by flow cytometry(BECKMAN COULTERDxFLEX).(9) Analyze the collected data using FlowJo-v10.8.1 software