Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS reprograms the lineage commitment of MSCs after GC treatment and inhibits the generation of primary senescent adipocytes. ( A ) Schematic illustration of the in vitro investigation of SCS targeting the prostaglandin/PPARγ/INK positive feedback loop in MPS-induced primary senescent adipocytes. ( B ) Representative flow cytometry plot showing p16 + senescent cells in adipocytes derived from bone marrow after 14 days of in vivo MPS induction and subsequently treated with SCS in vitro . ( C ) qPCR analysis of 12 senescence-associated markers in primary senescent adipocytes after in vitro SCS treatment. n = 3 biological replicates. ( D ) ELISA analysis of IL-1β levels in adipocyte supernatant following in vitro SCS treatment. n = 6 biological replicates. ( E ) ELISA analysis of secreted prostaglandins PGD2 and PGE2 in adipocytes under different treatment conditions. D-PBS: bone marrow adipocytes isolated from mice treated in vivo with the solvent control DMSO, followed by in vitro treatment with PBS; M-PBS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with PBS. M-SCS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with SCS. ( F ) Western blot analysis of intracellular COX-2 protein levels in adipocytes across the three treatment conditions. ( G ) Schematic illustration of competitive osteogenic–adipogenic differentiation of CD45 − Ter119 − CD31 − LepR + MSCs after 7 days of in vivo SCS and MPS co-treatment. ( H ) qPCR analysis of pan-adipocyte markers ( Fabp4 , Adipoq , Plin1 , Cd36 , and Lep ) in CD45 − Ter119 − CD31 − LepR + MSCs after 14 days of in vitro competitive lineage differentiation. n = 3 biological replicates. ( I and J ) Representative immunofluorescence images (I) and quantification (J) of perilipin + adipocytes and osteopontin + mature osteoblasts derived from lineage-committed MSCs. n = 6 biological replicates. (Scale bars, 30 μm, 15 μm and 15 μm). ( K ) Western blot analysis of adipogenesis-related markers C/EBPα, PPARγ, and C/EBPβ in the lineage-mixed cells after in vitro competitive differentiation of CD45 − Ter119 − CD31 − LepR + MSCs. ( L ) qPCR analysis of lipogenesis-related markers Fasn , Scd1 , Srebf1 , Acaca , and Acacb . n = 3 biological replicates. ( M and N ) Representative H&E staining images (M) of the femurs at day 14 following SCS and MPS co-treatment. Yellow arrows indicate bone marrow adipocytes. Magnified images show hypertrophic adipocyte morphology, with quantification of adipocyte diameter (N). n = 19 biological replicates. (Scale bars, 200 μm, 50 μm and 20 μm). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( C, D, H, J, L and N ), or one-way ANOVA with Tukey's post hoc test ( E ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: In Vitro, Flow Cytometry, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Solvent, Control, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Gene expression profiles of bone marrow-derived LepR + MSCs after 7-day in vivo co-treatment with SCS and MPS. ( A ) Heatmap showing DEGs in CD45 − Ter119 − CD31 − LepR + MSCs sorted from bone marrow at day 7 post-treatment with SCS versus PBS ( P < 0.05, |log fold change| > 1.5). n = 3 biological replicates. ( B ) Representative GO biological process enrichment analysis of downregulated DEGs. ( C ) Top 20 enriched KEGG pathways of downregulated DEGs in SCS versus PBS. ( D ) GSEA plots of biological processes positively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25). ( E ) Representative downregulated DEGs associated with adipogenesis and lipogenesis identified through KEGG pathway analysis. n = 3 biological replicates. ( F ) Top 20 enriched KEGG pathways of upregulated DEGs in SCS versus PBS. ( G ) Representative GO biological process enrichment analysis of upregulated DEGs. ( H ) Representative upregulated DEGs identified through biological process enrichment analysis. n = 3 biological replicates. ( I and J ) GSEA plots of KEGG pathways negatively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Gene Expression, Derivative Assay, In Vivo

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: CD45 antibody, anti-human, Vio R667, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-128-742 RRID: AB_2905022.

Techniques: Imaging, Comparison, Selection, Staining

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