Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 7 The number of T cells migrating across the transwell and the protein expression levels of chemokines mediating T-cell recruitment. (A) The activat ed CD3+ T cells were seeded into transwells with simple media and conditioned media from T24 cell cultures, NK cell cultures, and NK and T24 cell cocul tures. Summary data of the cumulative numbers of CD3+ T cells that migrated across the transwell in each condition are shown (n = 3 per group). ns, not significant; *p < 0.05 ; (B) Protein expression levels of chemokines in the supernatant from T24 culture, NK culture, and NK + T24 coculture measured using ELISA (n = 3 per group). Data expressed as mean ± SD were plotted. Abbreviations: CCL: C-C-motif ligand; CXCL:C-X-C-motif ligand; XCL1:X-C-motif ligand

Article Snippet: For chemokine release assay in transwell experiment, concentrations of human chemokines in the supernatant including C-C-motif ligand 1 (CCL1), CCL2, CCL4, CCL20, C-XC-motif ligand 1 (CXCL1), CXCL2, CXCL3, CXCL8, CXCL16, and C-motif ligand 1 (XCL1) were measured using human ELISA kits from CUSABIO for CCL1 (#CSB-EL004774HU), CCL2 (#CSB-Eq.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 8 Receptors of the chemokines expressed on CD3+ T cells and T cell count migrating across the transwell after blocking CCL1/2/20 in the superna tant of coculture medium. (A) The expressions of the chemokine receptors on CD3+CD56− T cells were detected using flow cytometry (n = 3 for all mol ecules). The representative images are shown. (B) Summary data of the cumulative numbers of T cells determined using a fluorescence cell analyzer after being harvested from the lower chambers, which were added into blocking antibodies against CCL1 (0.25 µg/mL), CCL2 (40 ng/mL), or CCL20 (10 µg/ mL) (n = 3 for each). Data are shown as mean ± SD. Statistical significance was determined using an unpaired t-test. *p < 0.05. Abbreviations: CXCR: C-X-C chemokine receptor; CCR:C-C chemokine receptor; XCR:X-C chemokine receptor

Article Snippet: For chemokine release assay in transwell experiment, concentrations of human chemokines in the supernatant including C-C-motif ligand 1 (CCL1), CCL2, CCL4, CCL20, C-XC-motif ligand 1 (CXCL1), CXCL2, CXCL3, CXCL8, CXCL16, and C-motif ligand 1 (XCL1) were measured using human ELISA kits from CUSABIO for CCL1 (#CSB-EL004774HU), CCL2 (#CSB-Eq.

Techniques: Cell Counting, Blocking Assay, Flow Cytometry, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: ODN 2088 modulates the release of chemokines by SC astrocytes, in vitro. a Representative chemokine arrays used to detect chemokines in CM of vehicle- and ODN 2088-treated astrocytes. The chemokine arrays were independently repeated twice, showing similar results. Results from a representative experiment are shown. The dots enclosed in rectangular boxes show chemokines whose levels were decreased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to the CM of vehicle-treated astrocytes. The dots enclosed in the oval box show the chemokine whose levels were increased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to CM of vehicle-treated astrocytes. 1: CCL1; 2: CCL9/MIP-1γ; 3: CCL2/MCP-1; 4: CCL20/MIP-3α; 5: CX3CL1. b Densitometric quantification of the signal obtained in the chemokine array using the Image Lab software (Bio-Rad). c Quantification of CCL9 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [** p < 0.01, independent-sample t -test, two-tailed]. The experiment was independently repeated four times, and the mean of 4 experiments ( n = 4) is shown. d Quantification of CCL2 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [* p < 0.05, independent-sample t -test, two-tailed]. The experiment was independently repeated three times, and the mean of 3 experiments ( n = 3) is shown

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: In Vitro, Software, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: CCL1 released by ODN 2088-treated astrocytes mediates the chemotaxis of peritoneal macrophages. Quantification of F4/80 + cells that crossed to the lower surface of the membrane in response to CM derived from vehicle- or ODN 2088-treated TLR9 astrocytes in the absence or presence of CCL1 neutralizing antibody or IgG 2A isotype control [ F (5, 48) = 81.03, p < 0.0001 by one-way ANOVA, **** p < 0.0001 by Tukey’s post hoc test]. The results of three independent experiments ( n = 3) are shown. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay, Membrane, Derivative Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: Astrocyte-derived CCL2 and CCL9 but not CCL1 regulate macrophage polarization, in vitro. a Macrophage cultures were exposed to ODN 2088-treated astrocyte CM (ODN 2088-CM), in the absence or presence of CCL1 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [ p = 0.7228, independent-sample t -test, two-tailed]. b Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL2 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [** p < 0.01, independent-sample t -test, two-tailed]. c Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL9 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [*** p < 0.001, independent-sample t -test, two-tailed]. d Macrophage cultures were exposed to vehicle-treated astrocyte CM (Veh-CM) or ODN 2088-treated astrocyte CM (ODN 2088-CM) for 24 h, with or without (control) addition of rmCCL9 (20 pg/ml). The graph shows the quantification of the F4/80 + /Arg-1 + double-labeled cells expressed as percent of total F4/80 + cells in macrophage cultures [ F (2, 6) = 53.68, p < 0.0001 by one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 by Tukey’s post hoc test]. The experiments were independently repeated twice, yielding similar results. Results from a representative experiment are shown. Results obtained from additional biological repeats of these experiments can be found in Additional file D-G. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Derivative Assay, In Vitro, Two Tailed Test, Control, Labeling

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: A scheme summarizing the effects of ODN 2088-treated astrocytes on macrophages. TLR9 antagonism increases the release of CCL1 by astrocytes, which enhances macrophage chemotaxis. In contrast, CCL2 and CCL9 release are decreased in response to ODN 2088. This reduces the negative regulatory effect of CCL2 and CCL9 on M2 macrophage polarization and fosters the M2 phenotype

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay

Journal: The American journal of pathology

Article Title: Chemokine (C-C Motif) Ligand 1 Derived from Tumor-Associated Macrophages Contributes to Esophageal Squamous Cell Carcinoma Progression via CCR8-Mediated Akt/Proline-Rich Akt Substrate of 40 kDa/Mammalian Target of Rapamycin Pathway.

doi: 10.1016/j.ajpath.2021.01.004

Figure Lengend Snippet: Figure 1 Up-regulation of chemokine (C-C motif) ligand 1 (CCL1) expression in tumor-associated macrophage (TAM)elike macrophages. A: Expression of CCL1 mRNA in peripheral blood monocyte (PBMo)ederived macrophages, TAM-like macrophages (TAM8, TAM9, and TAM15), and TE-8, TE-9, and TE-15 cells was determined using quantitative real-time PCR. Data were normalized to GAPDH levels (internal control). Assays were performed in triplicate. B: CCL1 con- centration in the supernatant of PBMo-derived macrophages, TAM-like macrophages (TAM8, TAM9, and TAM15), and TE-8, TE-9, and TE-15 cells. Protein levels were measured using an enzyme-linked immunosorbent assay. Assays were performed in triplicate. C: Expression of CCL1 in PBMo-derived macrophages and TAM-like macrophages (TAM8, TAM9, and TAM15) was confirmed by immunofluorescence using an anti-CCL1 antibody (green). Nuclei were stained with DAPI (blue). Macrophages were stained using an anti-CD204 antibody (red). Data are expressed as means SEM (A and B). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars Z 50 mm (C).

Article Snippet: After 48 hours, the supernatants were harvested, and CCL1 levels were determined using the Human I-309 ELISA Kit (CCL1) (number ab100536; Abcam), according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: The American journal of pathology

Article Title: Chemokine (C-C Motif) Ligand 1 Derived from Tumor-Associated Macrophages Contributes to Esophageal Squamous Cell Carcinoma Progression via CCR8-Mediated Akt/Proline-Rich Akt Substrate of 40 kDa/Mammalian Target of Rapamycin Pathway.

doi: 10.1016/j.ajpath.2021.01.004

Figure Lengend Snippet: Figure 3 Chemokine (C-C motif) ligand 1 (CCL1), secreted by tumor-associated macrophage (TAM)elike macrophages, promoted TE-8, TE-9, and TE-15 cell migration and invasion. Transwell migration and invasion assays were performed to confirm the effect of neutralizing antibodies against CCL1 or CCR8 on co-culturing TE-8, TE-9, or TE-15 cells with TAM-like macrophages (TAM8, TAM9, or TAM15, respectively). A: Cells in the upper chamber were treated with a neutralizing antibody against CCR8 (anti-CCR8; 10 ng/mL) or rat IgG (10 ng/mL; control IgG; negative control); migrated cells were counted after 24 hours. B: Neutralizing antibody against CCL1 (anti-CCL1; 0.2 mg/mL) or mouse IgG (0.2 mg/mL; control IgG; negative control) was added to the lower chamber; migrated cells were counted after 24 hours. C: Cells in the upper chamber were treated with anti-CCR8 (10 ng/mL) or rat IgG (10 ng/mL; control IgG; negative control); invaded cells were counted after 48 hours. D: Anti-CCL1 (0.2 mg/mL) or mouse IgG (0.2 mg/mL; control IgG; negative control) was added to the lower chamber; invaded cells were counted after 48 hours. Results are expressed as means SEM (AeD). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: After 48 hours, the supernatants were harvested, and CCL1 levels were determined using the Human I-309 ELISA Kit (CCL1) (number ab100536; Abcam), according to the manufacturer’s instructions.

Techniques: Migration, Control, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CX 3 CL1 Recruits NK Cells Into the Central Nervous System and Aggravates Brain Injury of Mice Caused by Angiostrongylus cantonensis Infection

doi: 10.3389/fcimb.2021.672720

Figure Lengend Snippet: The expression of CX 3 CL1 in the brain tissue and CX 3 CR1 on the CNS-infiltrated NK cells were elevated after A. cantonensis infection. The gene (A–G) and protein levels (H–N) of various chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, CXCL10 and CX 3 CL1) in brain tissue were measured by qRT-PCR and ELISA. (O–V) The gene expression levels of chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR8, CXCR3 and CX 3 CR1) on NK cells were detected by qRT-PCR. Multiple comparisons were performed by ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001. sNK, NK cells in spleen; bNK, NK cells in brain.

Article Snippet: And then, the levels of cytokines (IL-1β, IL-6 and TNF-α) and chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, CXCL10 and CX 3 CL1 in brain tissue homogenate samples were determined with commercial ELISA Kits (MultiSciences, Hangzhou, China) according to the manufacturer’s manuals.

Techniques: Expressing, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Gene Expression