Figure 1.

Differential protein surface expression and cell mechanics of immature and mature moDCs. moDCs were generated from monocytes of healthy donors within seven days in cell culture in the presence of GM-CSF and IL-4. On day 6 half of the cells were activated using a maturation cocktail consisting of PGE₂, TNFα, IL-1β, and IL-6. On day 7 immature (iDC) and mature (mDC) moDCs were harvested and 5 × 10⁵ cells were stained for FACS analysis with the antibodies CD11c-PE/Cy7 (1:100), HLA-DR-BV605 (1:100), CD83-A647 (1:100), and CD86-PE (1:100), or respective isotype controls. Cells were recorded using BD LSRFortessa and analyzed using FlowJo software. (A) Gating of double positive CD11c⁺HLA-DR⁺ immature and mature moDCs. Depicted is the gating of one representative donor. (B) Paired scatter plots of expression of activation markers CD83 and HLA-DR on immature and mature moDCs as well as the DC markers DEC205 and DC-SIGN. Median fluorescence intensity ΔMFI results of the MFI of antibodies against CLRs minus MFI of the corresponding isotype controls. Statistical significances were calculated using paired t-test (n≥10). Not significant (n.s.) p>0.05, *p<0.05, **p ≤ 0.01, ***p ≤ 0.001. (C–E) Real-time fluorescence deformability cytometry of immature (iDC) and mature (mDC) moDCs. moDCs were generated as described before. On day 7, immature and mature moDCs were harvested and 1 × 10⁶ cells were stained for real-time fluorescence deformability cytometry analysis with the antibodies CD83-PE (1:100) and HLA-DR-FITC (1:50). Real-time fluorescence deformability cytometry of immature and mature moDCs from one representative donor (n = 4). (C) Phase-contrast images of one representative immature and mature cell with contour detection showing deformation of moDCs. (D) Young’s modulus with (elastic modulus E) of immature and mature moDCs of the same representative donor as in (C). (E) Paired scatter plots of deformation, Young’s modulus and cell size (area) of all donors. Statistical significances were calculated using linear mixed model (n = 4). Not significant (n.s.) p>0.05, *p<0.05, **p ≤ 0.01, ***p ≤ 0.001.