Journal: Frontiers in Immunology

Article Title: Dendritic Cell Migration to Skin-Draining Lymph Nodes Is Controlled by Dermatan Sulfate and Determines Adaptive Immunity Magnitude

doi: 10.3389/fimmu.2018.00206

Figure Lengend Snippet: Skin distribution and ex vivo intrinsic migration potential of dendritic cells (DCs). (A) MHCII (green) expressing DC density in skin of wildtype (DseWT) and Dse -deficient (DseKO) mice. Three and four distances (in micrometers) are shown. The shortest distance is used (e.g., 19.6 µm) as average distance between MHCII expressing DCs and shown in the bar diagram ( n = 3 mice, ≥40 cells/mouse analyzed throughout the whole tissue). (B) Total CD11c+ DCs that migrated from dermal sheets of ears (dorsal part) upon mechanical disruption obtained from DseWT and DseKO mice. Cells were allowed to migrate for 40 h either or not in the presence of CCL21 (250 ng/mL). (C) DCs were isolated from spleen and allowed to migrate for 2 h in the absence (control) or presence of 250 ng/mL CCL21 in a Transwell system. (B) Bars indicate mean ± SEM ( n = 3 mice, one ear per condition, * p ≤ 0.05). (C) Bars indicate mean ± SEM of 4–6 experiments performed in triplicate (each dot represents an individual mouse, * p ≤ 0.05).

Article Snippet: After purification, 50 × 10 3 cells were placed in the upper compartment of a 5.0 μm pore size Transwell (Corning Costar Corp., Corning, NY, USA) and allowed to migrate for 2 h in the absence (medium only) or presence of 250 ng/mL recombinant mouse CCL21 (R&D systems) to the lower compartment.

Techniques: Ex Vivo, Migration, Expressing, Disruption, Isolation, Control

Journal: Nature Communications

Article Title: The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II

doi: 10.1038/s41467-021-23654-3

Figure Lengend Snippet: a Chemotaxis of WT (white bar) and Lamtor1 −/− (black bar) BMDCs toward the indicated concentrations of CCL19 (upper) or CCL21 (lower) in a Transwell assay system (pore size, 5 μm). Data are representative of three experiments. b – f Motility of DCs in response to CCL19 in 3D collagen matrices. Movement of WT (upper) and Lamtor1 –/– (lower) BMDCs in response to CCL19 (5 μg/ml) in type I collagen gels (2 mg/ml) in a Zigmond chamber were observed at 1-min intervals by time-lapse video imaging. Velocities of DCs were determined using the ImageJ manual tracking software. Consecutive images of DC locomotion: time (h:min) is shown above each panel. Scale bar, 30 μm ( b ). Mean velocity of the leading edge ( c ) and uropod ( d ), the maximum length of a single cell ( e ), and percentage of the immotile period of the uropod ( f ) ( n = 40 WT cells and n = 40 KO cells). g MLC phosphorylation in WT and Lamtor1 −/− BMDCs. MLC phosphorylation was evaluated by western blotting with anti-p-MLC antibody (upper). Data are representative of three experiments. Protein concentration in SDS–PAGE gel bands was determined using ImageJ, and statistical analysis was performed (lower) ( n = 3). h Localization of LAMP1 + lysosomes in non-polarized and polarized DCs. Lamtor1 −/− DCs were stained with anti-LAMP1 (green), phalloidin (blue), and DAPI (white), and visualized by confocal microscopy. Representative images of a non-polarized cell (left) and a polarized cell (right). Scale bar, 10 μm (left). Percentage of the region of interest (ROI) of perinuclear and peripheral regions of non-polarized cells and polarized cells ( n = 30) (right). i Lamp1 protein level of WT and Lamtor1 −/− BMDCs. The amount of Lamp1 protein was evaluated by western blotting with anti-Lamp1 antibody (upper). Data are representative of three experiments. The protein concentration in SDS–PAGE gel bands was determined using ImageJ and statistical analysis was performed (lower) ( n = 3). Statistical analyses were performed by two-sided Student’s t -test ( a , g , i ) [means ± s.d.; *p < 0.05, ** p < 0.01, ***p < 0.005] or two-sided Mann–Whitney U test ( c – f , h ) [median; 25th and 75th percentiles; and minimum and maximum of a population excluding outliers; *p < 0.001, ** p < 0.0001; NS, not statistically significant].

Article Snippet: Reagents were obtained from the indicated suppliers: LPS, PMA, rapamycin, FITC-isomer I, fibronectin OVA (Grade VI) CFA (Sigma); 2,4-DNFB (Tokyo Chemical Industry); collagenase D (Roche); type I collagen (BD Biosciences); calcein-AM, CFSE (carboxyfluorescein diacetate succinimidyl ester), phalloidin-546 (Invitrogen); AcidiFluor-ORANGE (GORYO Chemical); Torin1 (Selleck); U0216 (Calbiochem); blebbistatin (Cayman Chemical); recombinant mouse CCL19, mouse CCL21, human MCP-1 (R & D Systems); mouse GM-CSF (Wako); and Evans blue (Nacalai Tesque).

Techniques: Chemotaxis Assay, Transwell Assay, Pore Size, Imaging, Software, Phospho-proteomics, Western Blot, Protein Concentration, SDS Page, Staining, Confocal Microscopy, MANN-WHITNEY