mouse il 10 elisa kit  (R&D Systems)

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 drives tumor-educated M2-like polarization of macrophages and promotes tumor-cell aggressiveness. (A) Workflow for generating TC-hMDMs and TC-BMDMs, circSMAD4 knockdown, and downstream functional assays. (B) RT–qPCR analysis of M1-associated markers (MHC-II [HLA-DRA in TC-hMDMs; H2-Ab1 in TC-BMDMs], NOS2, and CD86) and M2-associated markers (CD163, CD206, and ARG1) in TC-hMDMs and TC-BMDMs. (C) Representative flow-cytometry histograms for HLA-DR, iNOS, CD86, CD163, CD206, and ARG1 in TC-hMDMs. Gating strategy and marker thresholds were defined based on FMO controls (see ). (D) Flow-cytometry quantification of marker-positive cells in TC-hMDMs and TC-BMDMs. (E) ELISA of IL-10, TGF-β, and iNOS in culture supernatants. (F) CCK-8 assays of A549 and LLC cells. (G) Colony-formation assays of A549 and LLC cells with quantification. (H) Bioluminescence-based growth readouts of patient-derived LUAD organoids (PDO #1 and PDO #2) after co-culture with TC-hMDMs. (I) Immunoblot analysis of EMT-related proteins (E-cadherin, N-cadherin, Vimentin) in A549 and LLC cells. (J) Transwell migration and invasion assays of A549 and LLC cells with quantification. Scale bar, 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: For mouse experiments, mouse IL-10 was measured using the Mouse IL-10 ELISA Kit (R&D Systems, Cat# M1000B), and mouse TGF-β1 was measured using the Mouse TGF beta-1 ELISA Kit (Invitrogen, Cat# BMS608-4), following the manufacturers’ instructions.

Techniques: Knockdown, Functional Assay, Quantitative RT-PCR, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Derivative Assay, Co-Culture Assay, Western Blot, Migration

Journal: Redox Biology

Article Title: Nrf2 pathway mediates copper oxide nanoparticle-induced exacerbation of allergic asthma

doi: 10.1016/j.redox.2026.104180

Figure Lengend Snippet: Effects of CuONPs on allergic inflammation and oxidative stress in OVA-induced asthmatic mice. (A) Airway hyperresponsiveness assessed as total respiratory system resistance in response to methacholine challenge (10, 20, and 40 mg/mL). (B–F) Total and differential inflammatory cell counts in BALF. (G–L) IL-1β, IL-6, TNF-α, IL-4, IL-5, and IL-13 levels in BALF, measured by ELISA. (M and N) Total IgE and OVA specific IgE levels in serum. (O and P) MDA levels and SOD activity in lung tissue. Data are presented as means ± SD (n = 6 mice/group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus NC group. # p < 0.05, ## p < 0.01, and ### p < 0.001 versus OVA group.

Article Snippet: BALF was centrifuged at 300× g for 10 min at 4 °C, and the supernatant was stored for cytokine analysis using commercially available enzyme-linked immunosorbent assay (ELISA) kits to quantify IL-1β, IL-6, tumor necrosis factor (TNF)-α, IL-4, IL-5, and IL-13 (R&D Systems, Minneapolis, MN, USA; Cat. No. MLB00C, M6000B, MTA00B, M4000B, M5000, and M1300CB, respectively).

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Redox Biology

Article Title: Nrf2 pathway mediates copper oxide nanoparticle-induced exacerbation of allergic asthma

doi: 10.1016/j.redox.2026.104180

Figure Lengend Snippet: Effects of Nrf2 overexpression on allergic inflammation and oxidative stress in CuONP-exposed asthmatic mice. (A) Immunofluorescence analysis of lung tissue from mice administered PBS or AAV2/8-GFP via intratracheal instillation. (B) Airway hyperresponsiveness assessed as total respiratory system resistance in response to methacholine challenge (10, 20, and 40 mg/mL). (C–G) Total and differential inflammatory cell counts in BALF. (H–M) IL-1β, IL-6, TNF-α, IL-4, IL-5, and IL-13 levels in BALF, measured by ELISA. (N and O) Total IgE and OVA specific IgE levels in serum. (P and Q) MDA levels and SOD activity in lung tissue. Data are presented as means ± SD (n = 3 mice/group for panels A; n = 6 mice/group for panels B–Q). In panel B, ## p < 0.01 indicates significant differences between GFP-NC and GFP-OVA, and ∗∗ p < 0.01 indicates significant differences between GFP-OVA + CuONPs and Nrf2-OVA + CuONPs. For panels C–Q, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between AAV-GFP and AAV-Nrf2 within each condition.

Article Snippet: BALF was centrifuged at 300× g for 10 min at 4 °C, and the supernatant was stored for cytokine analysis using commercially available enzyme-linked immunosorbent assay (ELISA) kits to quantify IL-1β, IL-6, tumor necrosis factor (TNF)-α, IL-4, IL-5, and IL-13 (R&D Systems, Minneapolis, MN, USA; Cat. No. MLB00C, M6000B, MTA00B, M4000B, M5000, and M1300CB, respectively).

Techniques: Over Expression, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activity 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: 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: Levels of angiogenesis-associated factors, including VEGF (Neobioscience, EMC103.96) and PDGF-BB (R&D Systems, MBB00), were quantified in both bone marrow supernatants and serum using ELISA kits according to the manufacturer's instructions.

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 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: Levels of angiogenesis-associated factors, including VEGF (Neobioscience, EMC103.96) and PDGF-BB (R&D Systems, MBB00), were quantified in both bone marrow supernatants and serum using ELISA kits according to the manufacturer's instructions.

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: Schematic illustration of the senescence-regulatory mechanisms of the sulfated polysaccharide in the glucocorticoid-induced bone marrow microenvironment. Bone marrow senescence plays a critical role in the pathogenesis of osteonecrosis. Glucocorticoids act on bone marrow target cells—adipocytes—to initiate primary bone marrow senescence via triggering a positive feedback loop through the prostaglandin/PPARγ/INK signaling axis. Subsequently, these senescent adipocytes propagate SASP factors to adjacent healthy cells through paracrine signaling or direct cell–cell contact, leading to secondary senescence. Sulfated chitosan (SCS) reprograms the lineage commitment bias of LepR + MSCs by activating the IGF-1/PI3K/Akt/mTOR signaling cascade, suppressing adipogenic differentiation and lipid biosynthesis pathways. SCS attenuates the spread of primary adipocyte senescence into secondary senescence, limiting the progressive amplification of the senescence cascade. Ultimately, this strategy halts the onset of senescence-driven osteonecrosis at an early stage and preserves the functional stability of the bone marrow microenvironment.

Article Snippet: Furthermore, to explore the molecular mechanisms by which SCS regulates MSCs lineage bias, bone marrow supernatant was collected on day 7 following co-treatment with SCS and MPS, and ELISA assays for IGF-1 (R&D Systems, MG100) and BMP-2 (R&D Systems, DBP200) were performed as described above.

Techniques: Amplification, Functional 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 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: Furthermore, to explore the molecular mechanisms by which SCS regulates MSCs lineage bias, bone marrow supernatant was collected on day 7 following co-treatment with SCS and MPS, and ELISA assays for IGF-1 (R&D Systems, MG100) and BMP-2 (R&D Systems, DBP200) were performed as described above.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, 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: In addition, ELISA analyses of senescence-associated inflammatory factors (IL-1β, IL-18, TNF-α, CCL3 (Neobioscience, EMC010a.96), CXCL1 (R&D Systems, MKC00B-1), and IL-6 (Neobioscience, EMC004.96)) were conducted on day 28 following combined treatment with SCS and rapamycin and/or T0070907.

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