anti cxcl12 neutralizing antibody  (R&D Systems)

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) GSEA plots of “chemokine signaling pathway” and “chemotaxis” gene sets enriched in MAOB-OE PrSC cells compared with controls. ( B ) ELISA of CXCL12 secretion in the culture media of control and MAOB-KD PrSC cells ( n = 3). ( C ) Representative IHC images and corresponding Pearson’s correlation analysis of stroma-expressed MAOB and CXCL12 protein levels in a PC TMA ( n = 37). Scale bars, 100 μm. ( D ) Pearson’s correlation analysis of MAOB and CXCL12 mRNA levels in patient-derived cultured prostatic stromal cells (left, n = 20) and laser-capture microdissected breast tumor stroma (right, n = 53) from GSE34312 and GSE9014 datasets, respectively. Statistical analysis was performed using one-way ANOVA with Dunnett’s test in (B). Data represent means ± SEM. ** P < 0.01.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay, Cell Culture

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Western blot of Twist1 in control and MAOB-manipulated PrSC cells upon NAC (5 mM, 48 hours) or H 2 O 2 (40 μM, 24 hours) treatment. ( B ) ELISA of CXCL12 secretion in culture media of control and MAOB-OE PrSC cells treated with TWIST1 siRNA or NAC (5 mM, 48 hours) ( n = 3). ( C ) qPCR of CXCL12 in indicated PrSC cells upon NAC treatment (5 mM, 48 hours) or Twist1/ TWIST1 siRNA expression ( n = 3). ( D and E ) Determination of CXCL12 mRNA (D) and 0.7-kb promoter activity (E) in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( F ) Schematic diagrams of WT and mutated CXCL12 E-box/SBE-Luc constructs and determination of their activities in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( G ) Representative proximity ligation assay staining and quantitation of indicated Twist1-Smad interactions by per-nucleus fluorescence intensity in control and MAOB-OE PrSC cells. Smad antibody incubation alone served as negative control. Numbers of nuclei included for comparisons between groups are denoted. Scale bars, 50 μm. ( H ) Co-IP assays of indicated Twist1-Smad interactions in PrSC cells with coexpression of Twist1 and individual Smads. Immunoglobulin G (IgG) was used in IP as negative control. Ten percent input was blotted as positive control. ( I ) ChIP analysis of chromatin from control and MAOB-OE PrSC cells precipitated with anti-Twist1, anti-Smad4, or a control IgG, followed by qPCR probing the E-box/SBE-centric CXCL12 promoter region ( n = 3). ( J ) ChIP analysis of chromatin from PrSC cells precipitated with anti-Smad4 antibody and then reprecipitated with anti-Twist1 or a control IgG (re-ChIP), followed by qPCR probing the E-box/SBE-encompassing CXCL12 promoter sequence ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay, Construct, Proximity Ligation Assay, Staining, Quantitation Assay, Fluorescence, Incubation, Negative Control, Co-Immunoprecipitation Assay, Positive Control, Sequencing

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 PC cells in monoculture and coculture with indicated PrSC fibroblasts upon anti-CXCL12 (0.1 μg/ml) antibody treatment ( n = 3). ( B ) Quantitation of PC cells in coculture with indicated PrSC cells treated with rCXCL12 protein (50 ng/ml; n = 3). ( C ) Western blot of CXCR4 and CXCR7 in control and CXCR4-KD/CXCR7-KD PC cells. ( D and E ) Quantitation of control and CXCR4-KD/CXCR7-KD PC cells in coculture with indicated PrSC cells treated without (D) or with (E) rCXCL12 (50 ng/ml; n = 3). ( F ) Quantitation of PC cells in coculture with indicated PrSC cells treated with 10 nM AMD3100 ( n = 3). ( G ) Representative images and quantitation of PC-3 cell migration and invasion in coculture with indicated PrSC cells upon treatment with anti-CXCL12 antibody (0.1 μg/ml) or 10 nM AMD3100 ( n = 3). Scale bars, 200 μm. ( H ) Representative images and quantitation of PC cell migration in coculture with indicated PrSC cells treated with rCXCL12 (50 ng/ml; n = 3). Scale bars, 100 μm. ( I ) Phospho-antibody array analysis of PC-3 cells treated with indicated PrSC cell CM. All phosphoprotein levels were normalized to their total forms from a single array with six replicate spots, with significantly activated phosphoproteins (fold change > 1.5, P < 0.05) denoted. ( J ) Western blot of p-Src and p-JNK in control and CXCR4-KD PC-3 cells treated with indicated PrSC cell CM. ( K ) Western blot of p-Src and p-JNK in PC-3 cells treated with indicated PrSC cell CM plus rCXCL12 (50 ng/ml). ( L ) Quantitation of PC cells in coculture with indicated PrSC cells following pretreatment with 40 nM Src inhibitor 1 or 10 μM SP600125 for 24 hours ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Quantitation Assay, Western Blot, Control, Migration, Ab Array

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 cell proliferation in monoculture and coculture with control and MAOB-OE PrSC cells upon selegiline treatment (10 nM, 72 hours) ( n = 3). ( B ) BLI-based growth curves of Luc-tagged PC-3 tumors grown in the prostates of male NSG mice treated with selegiline at various doses (0.5, 2, and 10 mg/kg) or saline as a vehicle ( n = 5 per group). ( C ) BLI images of mice from each group at the end point. ( D ) Determination of tumor weights ( n = 5). ( E ) Determination of MAOA and MAOB enzymatic activities in mouse liver tissue from each group at the end point ( n = 3). ( F ) Representative images of H&E and IHC staining of tumor-expressed Ki-67, p-Src, and p-JNK and stroma-expressed αSMA and CXCL12 and their quantitation in tumor samples from each group ( n = 5). Scale bars, 100 μm. ( G ) Mouse body weights determined weekly ( n = 5). ( H ) Representative H&E images of mouse liver and kidney tissue from each group. Scale bars, 100 μm. ( I to L ) ELISA of ALT (I), AST (J), BUN (K), and creatinine (L) in mouse sera at the end point ( n = 5). ( M ) Schematic depicting stromal-derived MAOB activation of paracrine CXCL12-CXCR4/Src/JNK signaling through interplay between ROS-dependent Twist1 (via a HIF1α/VEGF-A/AKT/FOXO1 pathway) and TGFβ1/Smads to promote stromal-epithelial interactions for PC growth and progression. Statistical analysis was performed using one-way ANOVA with Tukey’s test in (A), (D) to (F), and (I) to (L) and two-way ANOVA with Tukey’s test in (B) and (G). Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Quantitation Assay, Control, Saline, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activation Assay

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Defects of HSPC niche composed by osteoblastic cells in Bmi1 KO mice. (A) 3 rd passage of bone-derived mesenchymal progenitor cells (BdMPC) from 4-week-old WT and KO mice treated in osteoblast induction medium containing β-glycerophosphate and ascorbic acid for 4 days before ALP staining. ALP + osteoblasts shown in red after ALP staining. (B) The percentage of ALP + cell area versus total cell growth area was counted. n=3 samples. (C) Alp gene transcription levels in bulk mRNA from 3 rd passage of BdMPCs treated with osteoblast induction medium for 4 days in (A). n=3 samples. (D) Gene transcription levels of niche factors, including macrophage colony-stimulating factor ( M-csf ), N-cadherin, osteopontin ( Opn ), Cxcl12 , Angiopoietin 1 ( Ang1 ), stem cell factor ( Scf ), Jagged 1 ( Jag1 ), Vcam1 , integrin β1 and β2 , in 3 rd passage of BdMPCs from 3-week-old WT and KO mice were tested by qPCR. n=3 samples. (E-F) Protein levels of cellular (E) and secreted (F) CXCL12 and Ang1 were measured in cellular protein lysates and condensed medium supernatant from 3rd passage of BdMPCs by WB, respectively. (G) WT and KO BM cells were cultured in conditioned medium for 48 h before flow analysis. Conditioned medium (CM): Mixture of 50% (v/v) fresh culture medium with 50% conditioned medium collected from WT or KO BdMPC culture. The bar graph in right panel shows frequencies of LSK cells in cultured BM cells. n=4 samples. (H) Representative H&E-stained tibial paraffin sections from WT and KO mice with intratibial injection of vehicle or CXCL12. n=4 mice. (I) Flow analysis of LSK cells in BM of mice in Fig. H. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD; Unpaired two-tailed Student t test in (B), (C) and (D). One-way ANOVA with Tukey's post-hoc test in (G) and (I).

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Derivative Assay, Staining, Cell Culture, Injection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Sirt1 overexpression in MPCs partially prevented bone loss and CXCL12 reduction in Bmi1 KO mice. (A) BdMPCs from 1-m-old WT mice were transfected with Bmi1-pcDNA3.1 or vector plasmid using electroporation; 48 h post transfection, Bmi1 and Sirt1 expression was detected using immunofluorescence (IF) staining. (B) IF staining for Bmi1 and CXCL12 protein expression by BdMPCs as in (A). (C) Coronal sections of µCT 3D reconstruction of tibial metaphyseal region and (D) analyses of trabecular bone volume (BV/TV) and number (Tb.N) of tibiae from litters of 6-week-old WT, Sirt1 transgenic (TG), Bmi1 knockout (KO) and double mutant (KO-TG) mice. n=6 mice/group. (E) IHC staining for Collagen type-I (Col-I) expression on paraffin sections of proximal tibiae and (F) analyses of Col-1 + area in total tissue area (Col-I + A/T.A). n=6 mice/group. (G) Cxcl12 gene transcription levels in 3 rd passage of BdMPCs from 6-week-old WT, TG, KO and KO-TG mice. n=3 mice/group. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD. One-way ANOVA with Tukey's post-hoc test.

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Over Expression, Transfection, Plasmid Preparation, Electroporation, Expressing, Immunofluorescence, Staining, Transgenic Assay, Knock-Out, Mutagenesis, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Dietary supplementation of resveratrol partially rescues osteogenic defects in Bmi1 KO mice. (A) Coronal sections of µCT 3D reconstruction of tibial metaphyseal region and (B) analyses of trabecular bone volume (BV/TV) in tibiae from 4-week-old WT and Bmi1 knockout (KO) mice fed with vehicle- or resveratrol (RV)-added diet for 3 weeks before sacrifice. n=6 mice/group. (C) ALP staining on paraffin sections of proximal tibiae. (D) Histomorphometric analysis of ALP + area in total tissue area (ALP + A/T.A). n=6 mice/group. (E) H&E-stained paraffin sections of tibiae and (F) total bone marrow cell numbers in two femora were counted. n=6 mice/group. (G) Cxcl12 gene transcription levels in 3 rd passage of WT and KO BdMPCs treated with vehicle or resveratrol. n=3 samples. (H) ALP staining of 3 rd passage of BdMPCs from 4-week-old WT and KO mice and treated with osteoblast induction medium plus vehicle or resveratrol for 4 days. ALP + cell (red) area was counted. (I) Alp gene transcription levels in bulk mRNA from 3 rd passage of WT and KO BdMPCs treated with vehicle or resveratrol. n=3 samples. (J) Gene transcription levels of niche factors, including Jagged 1 ( Jag1 ), N-cadherin, osteopontin ( Opn ), stem cell factor ( Scf ), Vcam1 , integrin β1 and β2 , in 3 rd passage of WT and KO BdMPCs treated with vehicle or resveratrol tested using qPCR. n=3 samples. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD. One-way ANOVA with Tukey's post-hoc test.

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Knock-Out, Staining

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Resveratrol dietary supplement partially restored hematopoiesis in Bmi1 KO mice. (A) Representative flow graphs showing gates for LSK cell populations in BM of 4-week-old WT and KO mice that were fed with vehicle- or resveratrol (RV)-added diet for 3 weeks before sacrifice. (B-C) Frequencies (B) and numbers (C) of LSK in total BM cells were counted by flow. (D) Flow gates for LSK cell populations in spleen of mice as in (A) and, (E) the frequencies of LSK cells analyzed by flow. (F) The frequencies of B (B220 + ), T (CD3e + ) and myeloid (CD11b + ) cells in BM of mice in (A) were tested by flow. (G) WB of Sirt1, Cxcl12 and β-actin in protein lysates of WT and KO BdMPCs treated with vehicle or RV for 24 h. (H-J) BM cells from 1-m-old WT mice were cultured in conditioned medium from WT, KO and RV-treated KO BdMPCs plus Ang1 blocking peptide or anti-CXCL12 neutralizing Ab for 48 h. The frequencies of (H) LSK, (I-J) Ki67 + or Annexin V + LSK in culture BM cells were analyzed by flow. n=4 samples. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD. One-way ANOVA with Tukey's post-hoc test.

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Cell Culture, Blocking Assay

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: SNI produces persistent elevation of CXCL12 in plasma and hippocampal perivascular spaces. ( A ) Cytokine array results showing changes in plasma cytokines and chemokines in sham (Sh) and in 3 d, 9 d after SNI groups. There was no difference in the positive control protein levels (white boxes) among the three groups. ( B, C ) Plasma CXCL12 expression in different groups of female (F) and male (M) mice. n = 3 mice/group. ( D ) ELISA results showing changes in plasma CXCL12 concentrations with time after SNI, compared to sham mice (n = 3-6 mice/group). ( E ) SNI induced upregulation of CXCL12 in bilateral hippocampi (n = 4-6 mice/group). I: ipsilateral hippocampus, C: contralateral hippocampus. ( F ) CXCL12 mRNA in bilateral hippocampi at different time points after SNI (n = 7-12 mice/group). ( G, H ) Expression of CXCL12 around the hippocampal sulcus but not in the CA1 area was upregulated from 1 d to 9 d after SNI compared to sham mice (n = 3-4 slices, 3 mice/group). The white dotted boxes in top images are magnified below. Scale bars: 100 µm (top) and 25 µm (below). n.s.: no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. sham group, two-way ANOVA with Bonferroni's post-hoc test used for C and one-way ANOVA with Bonferroni's post-hoc test used for D-G. ( I ) Multiple sets of images show the colocalization of CXCL12 (red) with the PVM marker CD68 (green), endothelial cell marker PECAM-1 (green), astrocyte marker GFAP (green) or pericyte maker CD13 (green) in sham and SNI 3d groups. Red arrows showing the images in I are magnified from the red boxes in H. Scale bar = 25 µm.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Marker

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: Blocking the CXCL12-CXCR4 pathway reverses SNI-induced cognitive impairment, PVMs increase, and gliosis in the hippocampus. ( A ) Experimental protocol showing that anti-CXCL12 neutralizing antibody (20 ng/200 µL, i.v.) or CXCR4 antagonist AMD3100 (200 µg/mL, 1 mg/kg, i.p.) or vehicle (Vehi) was applied 30 min before and daily after sham (Sh) or SNI for 9 successive days. On day 9 after the injection, memory function was analyzed with NORT, and mice were perfused for IF and FC. ( B ) Anti-CXCL12 neutralizing antibody or AMD3100 injection prevented SNI-induced decline in the recognition index but had no effect in sham mice (n =5-12 mice/group). ( C-F ) Number of PVMs (CD68 high ) and the IntDen of CD68 high , PECAM-1, CD11b, and GFAP in the hippocampus in indicated groups. Scale bar = 100 µm. n = 3 mice/group, 3-4 slices/mice. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle sham group, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. vehicle SNI group, one-way ANOVA with Bonferroni's posthoc test.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Blocking Assay, Injection

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: Intravenous injection of CXCL12 induces memory deficit and elevates circulating monocytes and plasma CXCL12. ( A ) CXCL12 (1.0 and 2.5 ng/mL, 200 µL) or same volume of vehicle (Vehi) was injected for successive 9 days via the tail vein of naïve mice, and memory function was analyzed by NORT. The blood and brain tissue were harvested for ELISA, FC, and IF after the behavioral test. ( B ) Effects of injection of vehicle or different dosages of CXCL12 on STM index are shown (6-9 mice/group). ( C ) ELISA results revealed that CXCL12 injection at 2.5 ng/mL but not 1.0 ng/mL for 9 days elevated plasma CXCL12 (3-4 mice/group). The plasma CXCL12 was measured 24 h after the last injection of CXCL12. ( D-J ) Effects of iv injection of CXCL12 at 1.0 and 2.5 ng/mL on circulating leukocyte subpopulations. n = 5-6 mice/group. ** P < 0.01, *** P < 0.001 vs. vehicle group, one-way ANOVA with Bonferroni's post hoc test (B, C, H, J) and two-way ANOVA with Bonferroni's post hoc test (E, F).

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, IV Injection

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: CXCL12 intravenous injection increases perivascular macrophages and PECAM-1 expression and gliosis in the hippocampus. ( A , B ) Number of PVMs (CD68 high ) and immune fluorescence intensities of CD68 high in the hippocampal perivascular space in vehicle and CXCL12 groups (n = 3 mice, 3-4 slices /mice). ( C, D ) Effect of vehicle or CXCL12 injection on the CD11b, GFAP, PECAM-1, and CXCL12 expression. n = 3-4 slices/mice, 3 mice/group. Scale bar = 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle group, one-way ANOVA with Bonferroni's post-hoc test.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Injection, Expressing, Fluorescence

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: Circulating monocytes and plasma CXCL12 are increased in patients with chronic pain and are correlated with cognitive decline. ( A ) Memory function accessed by MoCA in patients with chronic neuropathic pain (Chronic pain, n = 30) was lower than healthy controls (Contr, n = 40). (B-G) Percentages of monocytes ( B ), granulocytes ( C ), neutrophils ( D ), lymphocytes ( E ), eosinophils ( F ), and basophils ( G ) in various groups were determined by routine blood analysis. ( H ) Concentration of plasma CXCL12 in healthy control subjects and chronic pain patients are shown. ** P < 0.01, *** P < 0.001 vs. healthy control group, two-tailed Student's t- test. ( I ) Scatterplots showing that the percentage of circulating monocytes (Spearman rank correlation, R 2 = 0.1022, P = 0.007) and plasma CXCL12 concentration (R 2 = 0.2323, P < 0.0001) was negatively correlated with MoCA scores in healthy controls and patients with chronic pain.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Concentration Assay, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: CD62L dim Neutrophils Specifically Migrate to the Lung and Participate in the Formation of the Pre-Metastatic Niche of Breast Cancer

doi: 10.3389/fonc.2020.540484

Figure Lengend Snippet: CD62L dim neutrophils initially aggregate in the lung by expressing CXCR4 at high levels, (A) RT-PCR analysis of chemokine receptor expression in CD62L dim and CD62L hi neutrophils. β-Actin expression in CD62L hi neutrophils was assayed as a control. (B) MFI of CXCR4 expression on CD62L dim and CD62L hi neutrophils from tumor-bearing mice, as analyzed using flow cytometry. (C) Flow cytometry analysis of the chemotaxis of cells induced by rCXCL12 after a 1:1 mixture of CD62L dim and CD62L hi neutrophils were seeded in the upper chamber. (D) Fluorescence imaging of labeled neutrophils after rCXCL12 chemotaxis. (E) RT-PCR analysis of Cxcl12 expression in various organs of naïve mice and 2-week tumor-bearing mice. (F) Flow cytometry analysis of the proportion of neutrophils after chemotaxis using the lung tissue culture supernatant (LTCS) of cells from tumor-bearing mice. (G) Flow cytometry analysis of the number of CD62L dim neutrophils after chemotaxis using the LTCS of cells from tumor-bearing mice in the presence or absence of the anti-CXCL12 mAb. Data are presented as the mean ± SEM of one representative experiment. Similar results were obtained in three independent experiments. Unpaired Student’s t tests, ns, not significant. *p < 0.05 and ***p < 0.001. See also .

Article Snippet: Medium supplemented with recombinant murine CXCL12 (100 ng/mL, Peprotech, #250-20A), lung tissue culture supernatant (LTCS), or a CXCL12 neutralizing antibody (200 μg/mL, R&D Systems, #MAB310-SP, clone: 79014) was added to the remaining receiver wells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Flow Cytometry, Chemotaxis Assay, Fluorescence, Imaging, Labeling