Journal: Signal Transduction and Targeted Therapy

Article Title: Thromboxane receptor activation in dendritic cells mitigates sepsis by suppressing S100a8/a9-mediated neutrophil recruitment

doi: 10.1038/s41392-026-02592-w

Figure Lengend Snippet: TP ablation in DCs exacerbates CLP- and LPS-induced sepsis in mice. a Survival analysis of CLP-challenged TP flox/flox ( n = 20 mice) and TP flox/flox CD11c Cre ( n = 27 mice) mice. b , c Plasma concentrations of proinflammatory (IL-1β) and anti-inflammatory (IL-10) cytokines in CLP-challenged TP flox/flox and TP flox/flox CD11c Cre mice ( n = 8 mice per group). d Lung vascular permeability detected by EBD leakage in TP flox/flox and TP flox/flox CD11c Cre mice at 0, 1, and 3 days post-CLP ( n = 6 mice per group). Lung wet/dry weight ratio ( e ) ( n = 6–8 mice per group) and BALF protein levels ( f ) ( n = 6–8 mice per group) in lungs from TP flox/flox and TP flox/flox CD11c Cre mice at 0, 1, and 3 days post-CLP. g Quantitative analysis of neutrophil infiltration via measurement of MPO activity in lung tissue from TP flox/flox and TP flox/flox CD11c Cre mice at 0, 1, and 3 days post-CLP ( n = 6 mice per group). h Representative histological images of lungs from CLP-challenged (0, 1, and 3 days) TP flox/flox and TP flox/flox CD11c Cre mice. Scale bars, 50 μm. i Quantification of histology scores for lung injury in ( h ) (n = 6–9 mice per group). j Survival analysis of LPS-treated TP flox/flox ( n = 15 mice) and TP flox/flox CD11c Cre ( n = 15 mice) mice. k , l Plasma cytokine levels of proinflammatory (IL-1β) and anti-inflammatory (IL-10) markers in LPS-treated TP flox/flox and TP flox/flox CD11c Cre mice ( n = 8 mice per group). m Lung vascular permeability detected by EBD leakage in LPS-treated (0, 8, and 24 h) TP flox/flox and TP flox/flox CD11c Cre mice ( n = 6 mice per group). Lung wet/dry weight ratio ( n ) ( n = 6 mice per group) and BALF protein levels ( o ) ( n = 5–6 mice per group) in LPS-treated (0, 8, and 24 h) TP flox/flox and TP flox/flox CD11c Cre mice. p Quantitative analysis of neutrophil infiltration via measurement of MPO activity in lung tissue from LPS-treated (0, 8, and 24 h) TP flox/flox and TP flox/flox CD11c Cre mice ( n = 6 mice per group). q Histological analysis of lung sections from LPS-treated (0, 8, and 24 hours) TP flox/flox and TP flox/flox CD11c Cre mice. Scale bars, 50 μm. r Lung histological scores corresponding to ( q ) ( n = 4 mice at 0 h, 6–8 mice at 8 and 24 h). Statistical significance was evaluated via two-way ANOVA followed by Sidak’s test for multiple comparisons ( b ‒ g , i , k ‒ p , r ) or the log-rank test ( a and j )

Article Snippet: The DCs were purified via CD11c UltraPure microbeads (Miltenyi, Cat# 130-125-835), achieving a purity of > 85%, as determined via flow cytometry.

Techniques: Clinical Proteomics, Permeability, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Thromboxane receptor activation in dendritic cells mitigates sepsis by suppressing S100a8/a9-mediated neutrophil recruitment

doi: 10.1038/s41392-026-02592-w

Figure Lengend Snippet: TP deficiency enhances neutrophil recruitment by increasing S100a8/a9 expression. a scRNA-seq analysis of CD45 + immune cells isolated from the spleens of TP flox/flox and TP flox/flox CD11c Cre ( n = 2 mice per group) mice at 3 days post-CLP. b Dot plots showing the expression level of the canonical annotation marker used for cluster annotation. c Proportions of immune cell clusters in the spleens of TP flox/flox and TP flox/flox CD11c Cre mice. Percentages ( d ) and numbers ( e ) of neutrophils (CD45 + CD3 − Ly6G + ) in the spleens of TP flox/flox and TP flox/flox CD11c Cre mice at 0, 1, and 3 days post-CLP. ( n = 4–6 mice per group). f Schematic of the neutrophil transwell migration assay. CFSE-labeled neutrophils from bone marrow were placed in the upper chamber, whereas DCs from TP flox/flox or TP flox/flox CD11c Cre mice were seeded in the lower chamber. Neutrophil migration was assessed after 2 h (Created with BioRender.com). g Quantification of neutrophil migration toward DCs from TP flox/flox and TP flox/flox CD11c Cre mice at 0 and 3 days post-CLP. ( n = 6 mice per group). h The correlation between TP receptor expression in blood DCs and neutrophil percentages in patients with sepsis ( n = 12 [control] and n = 23 [sepsis]) was analyzed. i Volcano plot of differentially expressed genes in DCs between TP flox/flox and TP flox/flox CD11c Cre mice (p.adjust < 0.05, Log2FC ≥ 0.5). j Top 10 enriched GO pathways associated with DEGs in TP flox/flox CD11c Cre vs TP flox/flox DCs. The mRNA expression of S100a8 ( k ) and S100a9 ( l ) in primary DCs from the spleens of CLP-challenged mice ( n = 4–6 mice per group). Plasma concentrations of S100a8 ( m ) and S100a9 ( n ) in CLP-treated (0 and 3 days) TP flox/flox and TP flox/flox CD11c Cre mice ( n = 8 mice per group). o , p Correlation analysis between TP and S100A8/A9 expression in blood DCs from patients with sepsis ( n = 12 [control] and n = 17 [sepsis]). q Neutrophil migration assay using DCs from TP flox/flox and TP flox/flox CD11c Cre mice post-CLP (3 days), with or without paquinimod ( n = 6–9 mice per group). r Schematic illustration showing that the deletion of TP promotes DC-mediated neutrophil recruitment through increased S100a8/a9 expression (Created with BioRender.com). Statistical significance was evaluated via Spearman’s correlation coefficient ( h , o , p ) and two-way ANOVA, followed by Sidak’s test ( d , e ) or Tukey’s test ( g , k – n , q ) for multiple comparisons

Article Snippet: The DCs were purified via CD11c UltraPure microbeads (Miltenyi, Cat# 130-125-835), achieving a purity of > 85%, as determined via flow cytometry.

Techniques: Expressing, Isolation, Marker, Transwell Migration Assay, Labeling, Migration, Control, Clinical Proteomics

Journal: Signal Transduction and Targeted Therapy

Article Title: Thromboxane receptor activation in dendritic cells mitigates sepsis by suppressing S100a8/a9-mediated neutrophil recruitment

doi: 10.1038/s41392-026-02592-w

Figure Lengend Snippet: Deletion of S100a9 in DCs alleviates CLP-induced sepsis in mice. a Survival analysis of CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice ( n = 20–26 mice per group). b , c Plasma cytokine levels of proinflammatory (IL-1β) and anti-inflammatory (IL-10) cytokines in CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice ( n = 8 mice per group). d Representative flow plots showing the percentages of DCs in the spleens of CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice. Quantification of DC percentages ( e ) and absolute numbers ( f ) in the spleens of CLP-challenged mice from ( d ) ( n = 6–8 mice). g Representative flow plots showing the percentages of neutrophils in the spleens of CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice. Quantification of neutrophil percentages ( h ) and absolute numbers ( i ) in the spleens of CLP-challenged mice from ( g ) ( n = 6–8 mice). j Representative immunofluorescence images showing the expression of neutrophil elastase (NE) and Ly6G in lung sections from CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice. Scale bars, 50 and 25 μm. k Quantification of NE-positive neutrophils in the lungs of ( j ) (n = 7–8 mice per group). l Western blot analysis of citH3 (NET marker) in the lungs of CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice after CLP challenge. m Quantification of protein levels in ( l ) ( n = 7 per group). n Quantitative analysis of neutrophil infiltration via measurement of MPO activity in lung tissue from CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice ( n = 6 mice per group). o Lung vascular permeability detected by EBD leakage in CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice ( n = 6 mice per group). Lung wet/dry weight ratio ( p ) (n = 8 mice per group) and BALF protein levels ( q ) ( n = 8 mice per group) in CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice. r Representative histology images of lung tissue from CLP-challenged CD11c Cre , TP flox/flox CD11c Cre , S100a9 flox/flox CD11c Cre , and TP flox/flox S100a9 flox/flox CD11c Cre mice. Scale bars, 50 μm. s Lung histology scores from ( r ) ( n = 7–9 mice per group). t Schematic illustration showing that ablation of S100a9 abolishes the enhanced neutrophil recruitment driven by TP-deficient DCs (Created with BioRender.com). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s test for multiple comparisons ( b , c , e , f , h , i , k , m – q , s ) or the log-rank test ( a )

Article Snippet: The DCs were purified via CD11c UltraPure microbeads (Miltenyi, Cat# 130-125-835), achieving a purity of > 85%, as determined via flow cytometry.

Techniques: Clinical Proteomics, Immunofluorescence, Expressing, Western Blot, Marker, Activity Assay, Permeability

Journal: Signal Transduction and Targeted Therapy

Article Title: Thromboxane receptor activation in dendritic cells mitigates sepsis by suppressing S100a8/a9-mediated neutrophil recruitment

doi: 10.1038/s41392-026-02592-w

Figure Lengend Snippet: Blocking the S100a8/a9 receptor (TLR4) alleviated sepsis. a Survival analysis of CLP-challenged TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment ( n = 21–26 mice per group). b , c Plasma cytokine levels of proinflammatory (IL-1β) and anti-inflammatory (IL-10) cytokines in CLP-challenged TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment ( n = 8 mice per group). d Representative flow plots showing the percentages of DCs in the spleens of TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment following CLP challenge. Quantification of the percentages ( e ) and absolute numbers ( f ) of splenic DCs in ( d ) ( n = 7–8 mice per group). g Representative flow plots showing the percentages of neutrophils in the spleens of TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment following CLP challenge. Quantification of splenic neutrophil percentages ( h ) and numbers ( i ) in ( g ) (n = 6–8 mice per group). j Representative immunofluorescence images showing the expression of NE and Ly6G in lung sections from CLP-challenged TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment. Scale bars, 50 and 25 μm. k Quantification of NE-positive neutrophils in the lungs of ( j ) ( n = 6–8 mice per group). l Western blot analysis of citH3 in the lungs of TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment after CLP challenge. m Quantification of protein levels in ( l ) ( n = 7 mice per group). n Representative histology images of lung tissues from CLP-challenged TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment. Scale bars, 50 μm. o Lung injury score quantification based on histological evaluation of the data in ( n ) ( n = 4–6 mice per group). Lung wet/dry weight ratio ( p ) and BALF protein levels ( q ) in CLP-challenged TP flox/flox and TP flox/flox CD11c Cre mice with or without Resatorvid treatment ( n = 8 mice per group). r Schematic illustration showing that blockade of the S100a8/a9 receptor TLR4 negates the increased neutrophil recruitment induced by TP-deficient DCs (Created with BioRender.com). Statistical significance was evaluated via two-way ANOVA followed by Tukey’s test for multiple comparisons ( b , c , e , f , h , i , k , m , o – q ) or the log-rank test ( a )

Article Snippet: The DCs were purified via CD11c UltraPure microbeads (Miltenyi, Cat# 130-125-835), achieving a purity of > 85%, as determined via flow cytometry.

Techniques: Blocking Assay, Clinical Proteomics, Immunofluorescence, Expressing, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Thromboxane receptor activation in dendritic cells mitigates sepsis by suppressing S100a8/a9-mediated neutrophil recruitment

doi: 10.1038/s41392-026-02592-w

Figure Lengend Snippet: TP activation inhibits S100a8/a9 transcription via PKCδ/Stat1 signaling. a Venn diagram illustrating the transcription factors (TFs) predicted to regulate S100a8/a9 expression identified through SCENIC analysis and PROMO database screening. b Heatmap displaying the activity levels of 21 transcription factors in control and TP-deficient DCs. c Violin plots showing the mRNA expression levels of four TFs ( Rela , Stat1 , Stat5a , and Atf1 ) in control and TP-deficient DCs from scRNA-seq. d Quantification of Stat1 expression in DCs from TP flox/flox and TP flox/flox CD11c Cre mice ( n = 5 mice per group). e Efficiency of siRNA-mediated Stat1 knockdown in control and U-46619-treated DC2.4 cells ( n = 6 wells per group). Relative mRNA levels of S100a8 ( f ) and S100a9 ( g ) in control and U-46619-treated DC2.4 cells after Stat1 knockdown ( n = 6–8 wells per group). h Western blot analysis of Stat1 and phosphor-Stat1 in spleen DCs from TP flox/flox and TP flox/flox CD11c Cre mice after CLP challenge. Effects of SQ29548 ( i ) and U-46619 ( j ) on S100a8 and S100a9 expression in DCs. k Effects of fludarabine on S100a8 and S100a9 expression in DCs with or without U-46619 treatment. l Western blot analysis of PKCβ, phosphor-PKCβ, PKCδ, and phosphor-PKCδ in spleen DCs from TP flox/flox and TP flox/flox CD11c Cre mice after CLP challenge. m Effect of rottlerin on Stat1 phosphorylation in DCs with or without U-46619 treatment. n , o Coimmunoprecipitation analysis of the interaction of PKCδ with Stat1 in DCs. p Effects of U-73122 on PKCδ and Stat1 phosphorylation in DCs treated with or without U-46619. q Effects of YM-254890 on phosphorylated PKCδ and phosphorylated Stat1 levels in DCs treated with or without U-46619. r Schematic illustration summarizing the TP-mediated inhibition of S100a8/a9 transcription via the G αq /PKCδ/Stat1 signaling pathway (Created with BioRender.com). Statistical significance was evaluated by the Mann–Whitney U test ( d ) and two-way ANOVA followed by Tukey’s test for multiple comparisons ( e – g )

Article Snippet: The DCs were purified via CD11c UltraPure microbeads (Miltenyi, Cat# 130-125-835), achieving a purity of > 85%, as determined via flow cytometry.

Techniques: Activation Assay, Expressing, Activity Assay, Control, Knockdown, Western Blot, Phospho-proteomics, Inhibition, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: Video showing DCs crawling around or inside LVs in uninflamed murine ear skin. Intravital microscopy was performed on uninflamed ear skin of anesthetized Prox1-mOrange YFP-CD11c mouse. DCs (green) can be observed crawling around or inside LVs (red). The video was generated from 3D reconstructions of Z-stacks. Video specifications: 20× objective, 30-s intervals, and 10 frames/s (300-fold accelerated). The original length of the recording: 60 min. Scale bar, 50 μm. Video length: 16 s.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Intravital Microscopy, Generated

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: Video showing DCs crawling around or inside LVs in inflamed murine ear skin. Intravital microscopy was performed on CHS-inflamed ear skin of anesthetized Prox1-mOrange YFP-CD11c mouse. DCs (green) can be observed crawling around or inside LVs (red). The video was generated from 3D reconstructions of Z-stacks. Video specifications: 10× objective, 30-s intervals, and 10 frames/s (300-fold accelerated). The original length of the recording: 60 min. Scale bar, 100 μm. Video length: 16 s.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Intravital Microscopy, Generated

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: Flow cytometry–based analysis of uPAR, uPA, and plasminogen protein levels on dermal cell subsets in vivo . Mice were sensitized with 2% oxazolone on the belly on day 0 and challenged on day 5 by applying 1% oxazolone to the skin of one ear. Flow cytometry was performed on both ears, i.e., the CTR and the CHS-inflamed ear, 1 day later. (A) Depiction of the gating strategy used for the identification of LECs (CD45 − CD31 + podoplanin + ), blood endothelial cells (CD45 − CD31 + podoplanin - ), leukocytes (CD45 + CD31 − ), and other nonvascular stromal cells (CD45 − CD31 − ). (B–E) Representative FACS plots (top) and summary of the delta mean fluorescent intensity (ΔMFI; specific - isotype staining) values obtained (bottom) when analyzing the expression of uPAR, uPA, and plasminogen in (B) LECs, (C) blood endothelial cells (BECs), (D) leukocytes, and (E) other nonvascular stromal cells of CTR or CHS-inflamed skin. Data points from the same animal (i.e., with one CTR and one CHS-inflamed ear, n = 4–7 mice in total) are connected by a line. Red lines indicate the mean. Paired Student’s t test.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Flow Cytometry, In Vivo, Staining, Expressing

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: Loss of uPA or of its cell surface localization alters DC entry into lymphatics. Quantitative whole-mount analysis of endogenous DC positioning in the steady-state ear skin of WT, uPA mut , uPA −/− , and CCR7 −/− mice. (A) Top row: Representative confocal images from the four genotypes. DCs are identified as CD45 + CD11c + cells (yellow). Bottom row: Confocal images with orthogonal views provided for one selected DC in the upper row image. Scale bars: 50 μm. The number in each image indicates the DC tissue position with respect to the LV, as defined in B. (B) Schematic depiction of the three different types of DC tissue positionings: (1) interstitial space, (2) adherent to the outer surface of the LV, and (3) within the LV lumen. (C) Quantification of the percentage DCs colocalized with lymphatics (i.e., percentage of (2+3)/(1+2+3), as defined in B). (D) Quantification of the percentage DCs localized inside the LV lumen (i.e., percentage of (3)/(1+2+3), as defined in B). Data points from the same experiment (involving one mouse per genotype) are connected by a line. n = 4–10 mice per condition. Paired Student’s t test.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques:

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: Supplemental data to FITC painting experiments and analysis of LNs draining CHS-inflamed skin. (A) Schematic depiction of the experiment: FITC was applied to the ear skin in WT and uPA mut mice, and ear skin or ear-draining auricular LNs were collected for analysis after 24 h. (B and C) Analysis of dermal DC numbers. Ear skin was enzymatically digested, and single-cell suspensions were generated for flow cytometry–based analysis. (D and E) Gatings used to identify migratory DCs (CD11c + MHCII hi ), and, amongst those, FITC + DCs in single-cell suspensions generated from (D) enzymatically digested LNs and (E) undigested LNs. (F–I) Analysis of immobilized CCL21 and plasmin(ogen) in CHS-dLNs. A CHS response was induced in the ear skin of WT mice, and ear-draining auricular LNs were collected 24 h later. (F and G) Analysis of CCL21 levels in LNs draining CTR or CHS-inflamed ear skin. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. (F) Representative images of the immunofluorescent staining and of the AI-based tissue segmentation used for differentiating between the T cell zone and B cell follicles/SCS. Scale bar: 100 μm. (G) Quantification of CCL21 staining intensity of observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. (H) ELISA-based quantification of (H) plasminogen and (I) plasmin activity in LN protein extracts, generated after perfusing the mice with PBS. Pooled data from n = 6 mice per group are shown in H and I, Student’s t test. (J) Representative gating strategy used for the quantification of FITC + CD11c + cells in LN sections from WT and uPA mut FITC-painted auricular LNs in . CD11c + cells were identified based on CD11c-AF647 positivity. From the CD11c + population, FITC + cells were identified and subsequently quantified. Marker-negative cells (red color) served as negative control to check background staining and to set the fluorescence thresholds. Please note that the data points of the WT CTR group in C are identical to those shown in , as these extracts were prepared and measured simultaneously.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Generated, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Marker, Negative Control, Fluorescence

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: DC migration from skin into the LN parenchyma is enhanced in uPA mut mice. FITC was applied to the ear skin in WT and uPA mut mice, and ear-draining auricular LNs were collected for analysis after 24 h. (A–D) Flow cytometry–based quantification of DCs in single-cell suspensions of enzymatically digested LNs. Percentage (A and C) and absolute numbers (B and D) of all migratory DCs (A and B; CD11c + MHCII + ) and FITC + migratory DCs (C and D). Pooled data from two similar experiments are shown ( n = 16–19 mice per group). (E–G) . Immunofluorescence-based analysis of sections prepared from auricular LNs after FITC painting. (E) Representative whole-slide multiplex immunofluorescence images of WT and uPA mut LNs. Images on the far left: overall LN architecture and examples of regions of interest (yellow boxes) used for high-resolution analysis of the SCS and parenchymal compartments. Subsequent images (left to right): higher-magnification images showing a merge of FITC signal, CD11c (DCs) and LYVE-1 (LVs) staining, followed by single-channel views of FITC and CD11c. Images on the far right: AI-based segmentation maps of FITC⁺CD11c⁺ DCs in the SCS or parenchyma. Scale bar.: 100 μm. (F) Percentage of FITC + CD11c + DCs localized in the SCS or in the LN parenchyma, and (G) ratio of FITC + CD11c + DCs in the SCS vs. LN parenchyma in WT and uPA mut LNs. Pooled data from the analysis of 6–7 mice per genotype are shown. Each dot presents the average of 3–6 images analyzed per one mouse. ( H ) Comparison of the LN cellularity retrieved from enzymatically digested (“D”) or nondigested (“ND”) LNs. Data points belong to the experiments described in A–D; “D” and I–L, “ND”. (I–L) Flow cytometry–based quantification of DCs in single-cell suspensions generated not digested LNs. Percentage and absolute numbers of (I and J) all migratory DCs (CD11c + MHCII + ) and (K and L) FITC + migratory DCs. Pooled data from three similar experiments are shown ( n = 31–34 mice per group). Unpaired Student’s t test (all graphs).

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Migration, Flow Cytometry, Immunofluorescence, Multiplex Assay, Staining, Comparison, Generated