Cd3 percp cyanine 5

Splenic lymphocytes were centrifuged at 180×g for 10 min and the cells were collected. The cells were re-suspended in PBS to a density of 2×10⁵ cells/ml, and labelled with anti-mouse monoclonal antibodies, including CD3-PerCP-Cyanine5.5 (45-0031-82), CD4-fluorescein isothiocyanate (CD4-FITC; 11-0041-82), CD8-phycoerythrin (CD8-PE; 12-0081-81), CD11c-APC (17-0114-82), major histocompatibility complex (MHC) class II-FITC (11-5322-81), CD86-PE (12-0869-42), CD40-PE-Cyanine7 (25-0409-42), and IDO-APC (17-9477-41) (eBioscience, San Diego, CA, USA), and Annexin-V and PI (V13241; Invitrogen, Shanghai, China). For CD4 and CD8 T cell staining, cells were incubated with 1 µl of CD3-PerCP-Cyanine5.5, 2 µl of CD4-FITC, and 2 µl of CD8-PE for 25 min at room temperature in the dark. Apoptosis was evaluated by incubation with Annexin-V and PI for 30 min at room temperature. For DCs markers, cells were incubated with 1 µl of CD11c-APC, 2 µl of MHC class II-FITC, 2 µl of CD86-PE, and 2 µl of CD40-PE-Cyanine7 for 30 min at room temperature. Samples were detected using an LSR II (BD Biosciences, NJ, USA) flow cytometer and analyzed using the FlowJo software (TreeStar, NJ, USA) according to the manufacturer’s instructions.

Eleven patients with IgAN were treated with valsartan (80–160 mg/d; Beijing Novartis Pharma Ltd, Beijing, China). Twenty-one IgAN patients were treated with Rehmannia glutinosa acteoside (0.8 g/d; Sichuan Medo Pharmaceutical Stock Co., Ltd, Sichuan, China) combined with valsartan (80–160 mg/d; Beijing Novartis Pharma Ltd, China). Twenty-four hour urinary protein were quantitatively analyzed. Peripheral blood Th22 lymphocytes were analyzed by flow cytometry as CD3⁺CD4⁺IL-17⁻INF-γ⁻IL-22⁺ lymphocytes. CD3 (PerCP-Cyanine5.5; eBioscience, Vienna, Austria), CD4 (FITC; eBioscience), CCR4 (APC; eBioscience), CCR6 (PE-Cyanine7; eBioscience), and CCR10 (PE; BioLegend, CA, USA) on T-cells isolated from the blood of patients with IgAN, and IFN-γ (APC; eBioscience), IL-22 (PE; eBioscience), IL-17 A (PE-Cyanine7; eBioscience), and Ki67 (Alexa 700 MAB; BD Biosciences, CA, USA) were stained using the fixation/permeabilization concentrate kit (BD Biosciences) according to the manufacturer’s instructions and analyzed by flow cytometry.