Fitc rat anti human cd4

Cells were collected in phosphate-buffered saline (PBS) containing 1% FBS (v/v) and fixed/permeabilized using the Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer’s instructions. For mouse iTreg analysis, selected protein markers were stained with fluorescein isothiocyanate (FITC) rat anti-mouse CD4, phycoerythrin (PE) rat anti-mouse CD25, and Alexa Fluor 647 rat anti-mouse Foxp3 monoclonal antibodies (BD Biosciences). For human iTreg analysis, protein markers were stained with FITC rat anti-human CD4, PE mouse anti-human CD25, and Alexa Fluor 647 mouse anti-human Foxp3 monoclonal antibodies (Biolegend).

Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of psoriatic patients and healthy donors by Ficoll-Paque gradient centrifugation (17144003, Cytiva). Human CD4⁺ T cells were isolated using a CD4⁺ T cell isolation kit (Cat#480,009, BioLegend) following the manufacturer’s protocol. Peripheral blood was obtained from experimental mice, and mouse CD4⁺ T cells were isolated using a CD4⁺ T cell isolation kit (Cat#19,852, Stem cell) following the manufacturer’s protocol. The human and mouse CD4⁺ T cell purity was confirmed to be above 95% by flow cytometric analysis. Flow cytometry was performed using FACS Calibur (BD Biosciences) and Cytoflex (Beckman) devices. Data were analyzed using FlowJo software (Tree Star, Ashland, OR). Th17 cells were defined as CD4⁺ IL17⁺ cells, and Tregs were defined as CD4⁺CD25⁺ Foxp3⁺ cells.
The following antibodies were used: FITC rat anti-human CD4 (Biolegend), APC mouse anti-human CD4 (Biolegend), PE/Cyanine5 mouse anti-human CD25 (Biolegend), PE mouse anti-human IL-17 A (eBioscience), FITC rat anti-mouse CD4 (Biolegend), PE rat anti-mouse CD3 (Biolegend), PE rat anti-mouse IL-17 A (Biolegend), and APC rat anti-mouse CD25 (Biolegend).