Figure 3.

New protocol for CD209⁺ DC isolation/expansion and transcriptional analysis. (A) Diagram of steps involved in CD209⁺ DC isolation (Created with BioRender.com). PBMC were isolated from blood and CD3/CD19/CD56 (Lineage, LIN) cells were depleted by positive isolation. The LIN^- cells were plated and treated with a cocktail of 70 ng/mL (GMCSF) (PeproTech) and 50 ng/mL IL-4 (PeproTech) to spike the CD209 population. 48 hours after CD209⁺ DC were sorted by positive magnetic isolation. (B) Flow plot to assess the purity of the CD209⁺ cells. The frequency of CD209 cells was around 50%, which was enriched to >96% in the isolated CD209⁺ cells were positive. FMO gating is also shown. (C) Representative microscopic images (Leica inverted microscope 40X) showing the morphology of isolated CD209⁺ DC. Cells appear heterogeneous with some more round shape cells and some elongated cells (in the magnification). (D) qPCR displaying the relative gene expression of MMP9, MMP2, CLU, SNX1, SNX2, LAMP1, TLR4, TREM1, MMP14, NOX2 in isolated CD209⁺ DC from HC (n=5), PsA (n=5) and RA (n=5) patients. Data are expressed as relative expression to HC (represented as red dotted line) and was calculated with the 2^–ΔΔCt method. Data are represented as mean ± SEM and differences among groups were evaluated by Non-parametric One-way ANOVA, Kruskal-Wallis test *p < 0.05, **p < 0.01”. (E) Heatmap representing -ΔCt value from HC (n=5), PsA (n=5) and RA (n=5) RNA isolated from circulatory CD209⁺ DC.