anti human mouse cxcl12  (R&D Systems)

Journal: Redox Biology

Article Title: Hydrogen sulfide inhibits recruitment of monocyte-derived tumor associated macrophages in glioblastoma by downregulating CXCL12

doi: 10.1016/j.redox.2025.103866

Figure Lengend Snippet: Histologic sections of IDH wildtype glioblastoma with low H 2 S concentrations (A, D, and G) and high H 2 S concentrations (B, E, and H) were immunostained with antibody against a TAM marker CD163 (A and B), combination of antibodies against a pan-myeloid marker Iba1 (green, D and E) and resident microglia specific marker P2RY12 (red, D and E), and antibody against chemokine CXCL12 (G and H). There was an inverse correlation between tumor H 2 S concentrations and density of CD163-positive TAMs (C; n = 10, ∗P = 0.044, Spearman), percent of Iba1-positive, P2RY12-negative, peripherally derived TAMs (F; n = 10, ∗P = 0.043, Spearman), and density of CXCL12 expressing cells (I; n = 10, ∗P = 0.0005, Spearman). Scale bar, 50 μm.

Article Snippet: Following heat-induced citrate buffer antigen retrieval and peroxide treatment, paraffin sections of human or mice tumors were incubated at 4 °C overnight with the following antibodies: rabbit anti-human CD163 (1:200, ab87099, Abcam), rabbit anti-human/mouse CXCL12 (1:50, MAB350, R&D Systems), rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba1; 1:500, NBP2-19019, Novus Biologicals), or rabbit anti-human hypoxia-inducible factor-1 (HIF-1; 1:100, ab51608, Abcam).

Techniques: Marker, Derivative Assay, Expressing

Journal: Redox Biology

Article Title: Hydrogen sulfide inhibits recruitment of monocyte-derived tumor associated macrophages in glioblastoma by downregulating CXCL12

doi: 10.1016/j.redox.2025.103866

Figure Lengend Snippet: (A) Wildtype mice fed a diet containing H 2 S donor SG1002 (40 mg/kg/d; n = 5) demonstrated increased plasma free H 2 S concentrations compared to those fed a control diet (n = 5; ∗∗∗∗P < 0.0001, t -test). (B) Similarly, relative plasma SSP4 fluorescence (indicating sulfane sulfur concentrations) was increased in the SG1002 group (n = 4) compared to controls (n = 4, ∗P = 0.016, t -test). (C) Kaplan-Meier curves showing overall survival for Nestin-TVA mice injected with RCAS-PDGFB and RCAS-STAT3 and fed a control (n = 25) or SG1002-containing diet (n = 25, ∗P = 0.020, log-rank test). Histologic sections of tumors from control (D, G, and J) and SG1002 groups (E, H, and K) were immunostained with an antibody against Iba1 (D and E), combination of antibodies against a pan-myeloid marker Iba1 (green, G and H) and resident microglia specific marker P2RY12 (red, G and H), and antibody against chemokine CXCL12 (J and K). SG1002 administration decreased accumulation of Iba1-positive TAMs (F, n = 4, ∗P = 0.013, t -test), relative abundance of Iba1-positive, P2RY12-negative, peripherally derived TAMs (I: n = 4, ∗∗P = 0.0018, t -test) and density of CXCL12 expressing cells (L, n = 3–4, ∗P = 0.036, t -test). Scale bar, 50 μm.

Article Snippet: Following heat-induced citrate buffer antigen retrieval and peroxide treatment, paraffin sections of human or mice tumors were incubated at 4 °C overnight with the following antibodies: rabbit anti-human CD163 (1:200, ab87099, Abcam), rabbit anti-human/mouse CXCL12 (1:50, MAB350, R&D Systems), rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba1; 1:500, NBP2-19019, Novus Biologicals), or rabbit anti-human hypoxia-inducible factor-1 (HIF-1; 1:100, ab51608, Abcam).

Techniques: Clinical Proteomics, Control, Fluorescence, Injection, Marker, Derivative Assay, Expressing

Journal: Redox Biology

Article Title: Hydrogen sulfide inhibits recruitment of monocyte-derived tumor associated macrophages in glioblastoma by downregulating CXCL12

doi: 10.1016/j.redox.2025.103866

Figure Lengend Snippet: (A) Transwell migration of THP-1 macrophages in response to conditioned media (CM) from T98G glioblastoma cells treated with PBS or 100 μM Na 2 S 4 , as well as serum-free media (NC) or media containing 10 % serum (PC). (B) CM from PBS treated T98G cells promoted chemotaxis of THP-1 macrophages compared to NC (∗∗P = 0.0070, ANOVA with Tukey's post hoc test), but this effect was abrogated with CM from Na 2 S 4 treated T98G cells. (∗P = 0.010, ANOVA with Tukey's post hoc test). Data are representative of 6 independent experiments per each condition. Scale bar, 50 μm. Na 2 S 4 treatment of T98G cells decreased CXCL12 transcript levels (C) and CXCL12 concentrations in CM (D) compared to PBS treatment (n = 3, ∗P = 0.010, ∗∗∗∗P < 0.0001, t -test).

Article Snippet: Following heat-induced citrate buffer antigen retrieval and peroxide treatment, paraffin sections of human or mice tumors were incubated at 4 °C overnight with the following antibodies: rabbit anti-human CD163 (1:200, ab87099, Abcam), rabbit anti-human/mouse CXCL12 (1:50, MAB350, R&D Systems), rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba1; 1:500, NBP2-19019, Novus Biologicals), or rabbit anti-human hypoxia-inducible factor-1 (HIF-1; 1:100, ab51608, Abcam).

Techniques: Migration, Chemotaxis Assay

Journal: Scientific Reports

Article Title: Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1

doi: 10.1038/s41598-025-16312-x

Figure Lengend Snippet: IHC of CXCL12 and α-SMA. ( A – D) CXCL12, ( E – H ) α-SMA, ( I – L ) Hematoxylin and eosin staining. Left two columns canine thyroid carcinoma tissue with a CXCL12 score of 3; Right two columns canine lung adenocarcinoma tissue with a CXCL12 score of 0. The left view of each case is at a low power field, and the right view is at a high-power field.

Article Snippet: For immunofluorescence (IF) analysis of fibroblasts markers, fibroblasts were seeded on chamber slides (eight-well NuncTM Lab-TekTM II Chamber SlideTM System; Thermo Fisher Scientific) at a density of 5.0 × 10 4 cells/well and cultured for 48 h. The cells were fixed with 4% paraformaldehyde at room temperature (RT) for 1 h, after which they were permeabilized with 0.5% Triton X-100 (Wako) in phosphate-buffered saline (PBS, Wako) for 15 min and blocked with 1% BSA for 1 h. Immunostaining was then performed using a rabbit anti-vimentin antibody (Clone SP20; 1:1000, Abcam, Cambridge, UK) as a marker for mesenchymal cells, a mouse anti-cytokeratin antibody (clone AE1/AE3; 1:200, Novus Biologicals, Centennial, CO, USA) as a marker of epithelial cells, a mouse anti-α-SMA (clone 1A4; 1:500, Thermo Fisher Scientific), and a mouse anti-CXCL12 antibody (clone 79,018; 1:40, R&D Systems), all diluted in PBS and incubated at 4 °C overnight.

Techniques: Staining

Journal: Scientific Reports

Article Title: Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1

doi: 10.1038/s41598-025-16312-x

Figure Lengend Snippet: Isolation and identification of fibroblasts. ( A ) Microscopic image of NFs and CAFs isolated from a dog with lung adenocarcinoma (optical microscope). Scale bars 50 µm. ( B ) IF staining for vimentin (red) and cytokeratin (green), and DAB staining for CD31 (brown) in NFs (top) and CAFs (bottom) isolated from a dog with lung adenocarcinoma. Nuclei were counterstained with DAPI (blue). Vimentin and cytokeratin were visualized using a confocal microscope, and CD31 staining was observed using an optical microscope. CD31 negativity confirmed the absence of endothelial cell contamination. Scale bars 50 µm. ( C ) IF staining for α-SMA and CXCL12 (green) in NFs (top) and CAFs (bottom). Nuclei were counterstained with DAPI (blue). Scale bars 50 µm (α-SMA), 20 µm (CXCL12).

Article Snippet: For immunofluorescence (IF) analysis of fibroblasts markers, fibroblasts were seeded on chamber slides (eight-well NuncTM Lab-TekTM II Chamber SlideTM System; Thermo Fisher Scientific) at a density of 5.0 × 10 4 cells/well and cultured for 48 h. The cells were fixed with 4% paraformaldehyde at room temperature (RT) for 1 h, after which they were permeabilized with 0.5% Triton X-100 (Wako) in phosphate-buffered saline (PBS, Wako) for 15 min and blocked with 1% BSA for 1 h. Immunostaining was then performed using a rabbit anti-vimentin antibody (Clone SP20; 1:1000, Abcam, Cambridge, UK) as a marker for mesenchymal cells, a mouse anti-cytokeratin antibody (clone AE1/AE3; 1:200, Novus Biologicals, Centennial, CO, USA) as a marker of epithelial cells, a mouse anti-α-SMA (clone 1A4; 1:500, Thermo Fisher Scientific), and a mouse anti-CXCL12 antibody (clone 79,018; 1:40, R&D Systems), all diluted in PBS and incubated at 4 °C overnight.

Techniques: Isolation, Microscopy, Staining

Journal: Scientific Reports

Article Title: Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1

doi: 10.1038/s41598-025-16312-x

Figure Lengend Snippet: Evaluation of CXCL12 and TGF-β1 secretion from CAFs. ( A ) CXCL12 concentrations in NF-CM and CAF-CM. Data are presented as mean ± SD (NF-CM, n = 7; CAF-CM, n = 10). ( B ) Scatter plot showing the correlation between CXCL12 levels in CAF-CM and serum CXCL12 concentrations, with Spearman’s rank correlation coefficient (rₛ) and P value indicated ( n = 7). ( C ) TGF-β1 concentrations in NF-CM and CAF-CM. Data are presented as median with IQR (NF-CM, n = 7; CAF-CM, n = 10). ( D ) CXCL12 secretion from CAFs cultured in the presence of the TGF-βRI inhibitor SB431542 ( n = 5). * P < 0.05, *** P < 0.001.

Article Snippet: For immunofluorescence (IF) analysis of fibroblasts markers, fibroblasts were seeded on chamber slides (eight-well NuncTM Lab-TekTM II Chamber SlideTM System; Thermo Fisher Scientific) at a density of 5.0 × 10 4 cells/well and cultured for 48 h. The cells were fixed with 4% paraformaldehyde at room temperature (RT) for 1 h, after which they were permeabilized with 0.5% Triton X-100 (Wako) in phosphate-buffered saline (PBS, Wako) for 15 min and blocked with 1% BSA for 1 h. Immunostaining was then performed using a rabbit anti-vimentin antibody (Clone SP20; 1:1000, Abcam, Cambridge, UK) as a marker for mesenchymal cells, a mouse anti-cytokeratin antibody (clone AE1/AE3; 1:200, Novus Biologicals, Centennial, CO, USA) as a marker of epithelial cells, a mouse anti-α-SMA (clone 1A4; 1:500, Thermo Fisher Scientific), and a mouse anti-CXCL12 antibody (clone 79,018; 1:40, R&D Systems), all diluted in PBS and incubated at 4 °C overnight.

Techniques: Cell Culture

Journal: Scientific Reports

Article Title: Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1

doi: 10.1038/s41598-025-16312-x

Figure Lengend Snippet: CAFs attract CD8⁺ T cells through the CXCL12/CXCR4 axis. ( A ) Schematic of the Boyden-chamber migration assay. CXCR4⁺CD8⁺ T cells were seeded in the upper compartment; the lower compartment contained control medium, recombinant CXCL12 (100 ng/mL) or CAF-CM. Migrated cells were counted after 24 h. ( B ) Migration toward recombinant CXCL12, expressed as fold change versus control. Mean ± SD ( n = 3). ( C ) Migration toward CAF-CM, expressed as fold change versus control. Mean ± SD ( n = 3). Control, culture medium; AMD3100, 4 µM CXCR4 inhibitor. ( D ) Schematic of the adhesion assay using a cell culture insert in a 6 cm dish. CAFs and tumor cells (RCM-SO or NMTCC) were plated on opposite sides of the insert and cultured for 24 h; the insert was then removed and CXCR4⁺ T cells added for a further 24 h. ( E ) Density of T cells adhered per cm 2 to cancer cell lines versus CAFs after 24 h. Mean ± SD ( n = 4). RCM-SO, canine mammary gland carcinoma cell line; NMTCC, canine transitional cell carcinoma cell line. ( F ) IHC of serial sections from a canine pulmonary adenocarcinoma stained for CXCL12 (top) and CD3 (bottom). Dashed lines delineate stromal and tumor cell areas, and asterisks (*) indicate the stromal regions. Scale bars 50 µm ( G ) CD3⁺ T cell density in stromal and tumor regions of thyroid carcinoma (TC, n = 2) and pulmonary adenocarcinoma (LC, n = 4). Numbers after histo-type indicate CXCL12 expression score. Mean ± SD. ( H ) Ratio of stromal to tumor CD3⁺ T cell density in each case. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For immunofluorescence (IF) analysis of fibroblasts markers, fibroblasts were seeded on chamber slides (eight-well NuncTM Lab-TekTM II Chamber SlideTM System; Thermo Fisher Scientific) at a density of 5.0 × 10 4 cells/well and cultured for 48 h. The cells were fixed with 4% paraformaldehyde at room temperature (RT) for 1 h, after which they were permeabilized with 0.5% Triton X-100 (Wako) in phosphate-buffered saline (PBS, Wako) for 15 min and blocked with 1% BSA for 1 h. Immunostaining was then performed using a rabbit anti-vimentin antibody (Clone SP20; 1:1000, Abcam, Cambridge, UK) as a marker for mesenchymal cells, a mouse anti-cytokeratin antibody (clone AE1/AE3; 1:200, Novus Biologicals, Centennial, CO, USA) as a marker of epithelial cells, a mouse anti-α-SMA (clone 1A4; 1:500, Thermo Fisher Scientific), and a mouse anti-CXCL12 antibody (clone 79,018; 1:40, R&D Systems), all diluted in PBS and incubated at 4 °C overnight.

Techniques: Migration, Control, Recombinant, Cell Adhesion Assay, Cell Culture, Staining, Expressing

Journal: Scientific Reports

Article Title: Exploring the effect of canine cancer-associated fibroblasts on T cell dynamics through the CXCL12/CXCR4 axis modulated by TGF-β1

doi: 10.1038/s41598-025-16312-x

Figure Lengend Snippet: Scheme of CAF-mediated regulation of T cell dynamics through the CXCL12/CXCR4 axis and TGF-β1 signaling. TGF-β1 secreted by CAFs may contribute to the upregulation of CXCR4 on T cells and enhance CXCL12 secretion from CAFs. T cells with increased CXCR4 expression could be attracted to CAF-derived CXCL12 within the TME. This mechanism may potentially interfere with T cell migration toward cancer cells and contribute to immune exclusion.

Article Snippet: For immunofluorescence (IF) analysis of fibroblasts markers, fibroblasts were seeded on chamber slides (eight-well NuncTM Lab-TekTM II Chamber SlideTM System; Thermo Fisher Scientific) at a density of 5.0 × 10 4 cells/well and cultured for 48 h. The cells were fixed with 4% paraformaldehyde at room temperature (RT) for 1 h, after which they were permeabilized with 0.5% Triton X-100 (Wako) in phosphate-buffered saline (PBS, Wako) for 15 min and blocked with 1% BSA for 1 h. Immunostaining was then performed using a rabbit anti-vimentin antibody (Clone SP20; 1:1000, Abcam, Cambridge, UK) as a marker for mesenchymal cells, a mouse anti-cytokeratin antibody (clone AE1/AE3; 1:200, Novus Biologicals, Centennial, CO, USA) as a marker of epithelial cells, a mouse anti-α-SMA (clone 1A4; 1:500, Thermo Fisher Scientific), and a mouse anti-CXCL12 antibody (clone 79,018; 1:40, R&D Systems), all diluted in PBS and incubated at 4 °C overnight.

Techniques: Expressing, Derivative Assay, Migration