Apc cy7 livedead

hPBMCs (Lonza, Basel, Switzerland) were cultured in a lymphocyte growth medium (LGM-3, Lonza) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Cells were seeded at a density of 1 million cells per well in a 24-well cell culture plate (SPL Life Sciences, Pocheon, Republic of Korea) and treated with ProLNG-001 at the indicated concentration. For cytokine measurement, cells were incubated for 24 h at 37 °C in a humidified 5% CO₂ incubator. After 24 h, the cell culture supernatants were collected, and the IFN-γ secretion was measured using the IFN-gamma Human Uncoated ELISA Kit (Thermo Fisher Scientific). For the analysis of IFN-γ-secreting CD8+ T cells, FACS analysis was performed. The cells were stimulated with a cell stimulation cocktail (plus protein transport inhibitors 0.5×, Thermo Fisher Scientific) for 6 h, after 48 h of incubation with the samples. After stimulation, the cells were intracellularly stained using the Intracellular Fixation & Permeabilization Buffer Set Kit (Thermo Fisher Scientific) following the manufacturer’s recommendations. The cells were stained with APC-Cy7-Live/Dead (Thermo Fisher Scientific), BV421-CD3, PE-CD8, and PerCP-Cy5.5-IFN-γ antibodies (BioLegend, San Diego, CA, USA).

For the flow cytometric analysis sorting, skin cells were sorting according to Propidium Iodide (Biosharp, China). The PI-negative cells were immunolabeled with IL-20RB Polyclonal antibody (Proteintech, USA), then immunolabeled with Goat Anti Rabbit IgG(H&L)-Alexa Fluor 488 and PE anti-human CD140b (PDGFRB) Antibody (Biolegend, USA). IL20RB-positive and PDGFRB-positive cells were collected on Beckman MoFloXDP for RT-qPCR analysis of gene expression levels, the result was analyzed by FlowJo.
For flow cytometry, live DCs were immunolabeled with APC-CY7-livedead (Thermo Fisher Scientific, Massachusetts, USA), Brilliant Violet 605™ anti-mouse CD11C (BioLegend, CA), PE anti-mouse CD80 (Thermo Fisher Scientific), FITC anti-mouse CD86 (Thermo Fisher Scientific) at 4°C for 30 min. All cells were detected on Beckman Cytoflex LX and the result was analyzed by FlowJo.

DCs were generated from the bone marrow cells of 8 weeks old female BALB/c mice. The bone marrow cells were centrifuged, washed and cultured in roswell park memorial institute 1640 medium (Hyclone, Utah, USA) containing 10% fetal bovine serum (Sciencell, CA, USA), 1% penicillin/streptomycin (Beyotime), 10 ng/ml recombinant human IL-4 protein (R&D systems, Minnesota, USA) and 20 ng/ml granulocyte macrophage-colony stimulating factor (PeproTech, NJ, USA) and incubated at 37 °C and 5% CO2 for seven days. Cells were cultured with recombinant human IL-17B protein (500 ng/mL) (R&D systems) for 24 h. Cells cultured with medium provided a negative control while cells cultured with LPS (10 ng/mL) (Sigma, Missouri, USA) provided the DC activation positive control. DC phenotypes were analyzed via surface expression of specific markers. Live DCs were immunolabeled with APC-CY7-livedead (Thermo Fisher Scientific, Massachusetts, USA), eFluor450-CD11C (Thermo Fisher Scientific), PE-CD80 (Thermo Fisher Scientific), APC-CD86 (Thermo Fisher Scientific), FITC-MHC II (Thermo Fisher Scientific) at 4 °C for 30 min. Resuspend cells were detected on Beckman Cytoflex LX and the result was analyzed by FlowJo 10.