Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Immunohistochemical preparations of adjacent sections of a D-12 tumour. An avidin-biotin peroxidase-based method was used for staining. Haematoxylin was used for counterstaining. IL-8 positive foci visualized by anti-IL-8 antibody staining ( A ) and hypoxic foci visualized by anti-pimonidazole antibody staining ( B ).

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques: Immunohistochemical staining, Avidin-Biotin Assay, Staining

Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Hypoxia, IL-8 expression and neovascularization in metastatic and non-metastatic D-12 primary tumours. Points represent single tumours. Density of hypoxic foci ( A ), density of IL-8 positive foci ( B ) and density of vascular hot spots ( C ).

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Effects of anti-VEGF treatment, anti-IL-8 treatment, and combined anti-VEGF and anti-IL-8 treatment on development of hypoxia, neovascularization and spontaneous pulmonary metastasis in D-12 tumours. ( A ) Percentage of mice with metastasis. Points represent single experiments involving 10 mice each. ( B ) Densities of hypoxic foci and vascular hot spots. Columns represent mean values of 20 mice. Bars represent s.e.m.

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques:

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter inoculation increased the secretion of IL-8 and TNF-α in polarized human intestinal epithelial T84 cells. T84 cells were cultured on transwells until the transepithelial resistance reached 1,400 Ω/cm2. C. jejuni 81-176 was inoculated from the apical chamber at an MOI of ∼10 and incubated at 37°C for 0, 4, 6, or 24 h. For the 24-h time point, the cells were washed at 6 h to remove unattached bacteria and cultured in fresh medium for another 18 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation at any time point. The supernatants from the apical and basolateral chambers were collected separately; the bacteria were removed by centrifugation; and protease inhibitors were added to prevent protein degradation. The concentrations of IL-8 (A) and TNF-α (B) were determined by ELISA. Averages for three independent experiments are shown; error bars, standard deviations. Asterisks represent significant (P < 0.05) differences from control cells that were not inoculated with the bacteria.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Cell Culture, Incubation, Centrifugation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-stimulated IL-8 secretion is not dependent on the secretion of TNF-α. (A) Polarized T84 cells were incubated with different concentrations of TNF-α (0, 5, 10, 50, and 100 ng/ml) basolaterally at 37°C for 24 h. The basolateral media were collected, and the concentrations of IL-8 were measured using ELISA. (B) IL-8 concentrations in the basolateral medium were measured using ELISA at 24 h after T84 cells were incubated under the following conditions: TNF-α (50 ng/ml) applied apically (Apical) (bar I), TNF-α applied basolaterally (Basol) (bar II), (III) TNF-α applied basolaterally plus mouse anti-human TNF-α antibody (5 μg/ml) applied both apically and basolaterally (Both) (bar III), or live C. jejuni 81-176 applied apically plus mouse anti-human TNF-α antibody applied both apically and basolaterally (bar IV). Averages from three independent experiments are shown; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Comparison of IL-8 secretion induced by different Campylobacter strains inoculated apically or basolaterally. (A and B) Polarized T84 cells were inoculated from either the apical (A) or the basolateral (B) side with chicken meat isolates (C. coli [C] or C. jejuni [J] strains) or with C. jejuni 81-176 or its flaA mutant at an MOI of ∼10 and were incubated for 24 h. At 6 h, T84 cells were washed and placed in fresh medium. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. Uninfected T84 cells were used as controls. The apical and basolateral media were collected separately at 24 h, and the concentrations of IL-8 were measured by ELISA. Averages for three independent experiments are shown; error bars, standard deviations. (C) Pearson's correlation coefficients between IL-8 secretion and Campylobacter adherence, invasion, and transcytosis efficiencies (expressed as percentages of the inocula) were calculated and tested for significance (α = 0.05 by a two-tailed test).

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Mutagenesis, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: IL-8 secretion induced by conditioned supernatants generated from Campylobacter-T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA, pflA, cdtB, or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P < 0.05. (F) The cytotoxicity of CDT was measured by incubating Vero cells with serially diluted C. jejuni culture supernatants that were filtrated and treated with polymyxin B for 48 h. The viability of the Vero cells was monitored using an MTT assay. The CDT titers were expressed as the reciprocal of the highest dilution that caused 50% Vero cell death compared with the level in untreated cells. Data are averages for three independent cytotoxicity assays; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Generated, Incubation, Cell Culture, Mutagenesis, Enzyme-linked Immunosorbent Assay, MTT Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-induced IL-8 secretion depends on Campylobacter-secreted CDT and the TLR signaling adaptor MyD88 in polarized intestinal epithelial cells. (A) Polarized T84 cells were pretreated with MyD88 inhibitory peptide (100 μM) (bars III, V, and VII) or its control peptide (bars II, IV, and VI) for 24 h or with chloroquine (5 μM) (bars VIII, IX, and XIII) for 30 min before being incubated with the conditioned supernatant (ConSup) from the coculture of T84 cells with wt C. jejuni 81-176 (bars II, III, and VIII), its cdtB mutant (bars IV, V, and IX), or a C. jejuni 81-176 DNA extract (25 μg/ml) (bars VI and VII) at 37°C for 24 h. MyD88 inhibitory peptide, its control peptide, or chloroquine was also included during the 24-h incubation. Polarized T84 cells incubated with TNF-α only (bar XII) or TNF-α plus chloroquine (bar XIII) were used as controls for the effect of chloroquine on IL-8 secretion. Polarized T84 cells incubated with the medium alone for 24 h (bars I) or with live C. jejuni 81-176 for 4 h (bars X), after which the conditioned supernatants were collected, or 24 h (bars XI) were used as controls. In the 24-h incubation with live bacteria, unbound bacteria were removed at 6 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. The apical and basolateral media were collected individually, and the IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. (B) Polarized T84 cells were pretreated with chloroquine (5 μM) for 30 min before incubation with C. jejuni 81-176 (MOI, ∼10) at 37°C for 4 h. The abilities of C. jejuni 81-176 to adhere to, invade, and transcytose across chloroquine-treated T84 cells were analyzed as described in the legend to Fig. ​Fig.3.3. Averages for three independent experiments with triplicate samples are shown; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Incubation, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-induced IL-8 secretion is dependent on NF-κB activation. (A) T84 cells were incubated with C. jejuni 81-176 (MOI, ∼10) in the presence of cycloheximide for varying lengths of time. T84 cells treated with TNF-α were used as a positive control. −, no treatment. The cells were washed and lysed, and the cell lysates were analyzed by SDS-PAGE and Western blotting with probing for IκB-α. The blots were stripped and reprobed for tubulin as a loading control. Representative results from three independent experiments are shown. (B) Polarized T84 cells were pretreated with the NF-κB inhibitor SN50 (18 μM), quinazoline (28 μM), or TPCK (50 μM) for 30 min and then incubated with C. jejuni 81-176 in the presence of the inhibitor at 37°C for 24 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. The apical and basolateral media were collected individually, and the IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Activation Assay, Incubation, Positive Control, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Cytokine arrays of 143B-Luc cells, human macrophages, and co-culture CM. B IL-8 production during the co-culture of OS cells (143B-Luc, SJSA-1) and macrophages (THP-1 Mφ, HMDMs). C IL-8 expression in macrophages stimulated with OS-CM. D IL-8 expression in OS cells stimulated with macrophage CM. E UMAP plot of OS lung metastases. F UMAP plot of Iba1, CD163, and IL-8 expression in myeloid cell clusters. G Violin plot depicting IL-8 expression in Iba1 +/− or CD163 +/− myeloid cell clusters. CM conditioned medium, HMDMs human monocyte-derived macrophages, OS osteosarcoma, THP-1 Mφ THP-1-derived macrophage. Data are presented as mean ± standard deviation. **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Co-Culture Assay, Expressing, Derivative Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Cell viability assay for OS cells treated with or without recombinant IL-8 (rIL-8, 10 ng/mL) using Cell Counting Kit-8. B , C Scratch and invasion assays for OS cells treated with or without co-culture CM. The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. D Cell viability assay for OS cells treated with or without co-culture CM and with or without anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) using Cell Counting Kit-8. E , F Scratch and invasion assays for OS cells treated with or without co-culture CM and with or without IL-8 Abs (100 ng/mL). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM: conditioned medium, OS: osteosarcoma, ns: not significant. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Viability Assay, Recombinant, Cell Counting, Co-Culture Assay, Wound Healing Assay, Invasion Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Western blot assays for FAK phosphorylation in OS cells treated with co-culture CM and quantification of western blot bands. B Western blot assays for the phosphorylation of FAK (p FAK) in OS cells treated with co-culture CM + isotype IgG (IgG) or + anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) and quantification of western blot bands. The FAK phosphorylation levels were also compared between the same times. C Cell viability assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186, 1 µM) using Cell Counting Kit-8. D , E Scratch and invasion assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186) (1 µM). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM conditioned medium, FAK focal adhesion kinase, ns not significant, OS osteosarcoma. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Western Blot, Co-Culture Assay, Viability Assay, Cell Counting, Invasion Assay, Wound Healing Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Schematic showing the experimental design for subcutaneous transplantation of OS cells. 143B-Luc cells (2 × 10 6 /mouse) were subcutaneously transplanted in 6–7-week-old BALB/c nu/nu mice, and DMEM or co-culture CM was injected into the para-tumor thrice per week. B Tumor volume with or without co-culture CM measured thrice per week after tumor transplantation. C , D Tumor weight and image of the excised tumor with or without co-cultured CM 14 days after tumor transplantation. E , F Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with or without co-culture CM 14 days after tumor transplantation. Two fields of view per sample were randomly selected, and quantification was performed in 10 fields. E Ki-67-positive cells in the field were counted and indicated as Ki-67 labeling index. Scale bars represent 50 μm. G Schematic showing the experimental design for subcutaneous OS transplantation using anti-IL-8 antibodies (IL-8 Abs). H Tumor volume after pretreatment with co-culture CM with or without IL-8 Abs (10 µg/mouse) measured thrice per week after tumor transplantation. I and J Weight and image of the excised tumor pretreated with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. K , L Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. Scale bars represent 50 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, FAK focal adhesion kinase, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Transplantation Assay, Co-Culture Assay, Injection, Cell Culture, Immunohistochemistry, Labeling, Modification, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Schematic showing the experimental design for OS tail vein injection. 143B-Luc cells were treated with DMEM or co-culture CM for 12 h prior to tail vein injection; 143B-Luc cells (1 × 10 6 /mouse) were injected into the tail vein of 6–7 week-old BALB/c nu/nu mice. B IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with or without co-culture CM. C Hematoxylin and eosin staining of lung sections with or without co-culture CM and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. D Immunohistochemistry of Ki-67 positive cells in lung colonies with or without co-culture CM and quantification of the Ki-67 labeling index. Two colonies per sample were randomly selected, and quantification was performed for 10 colonies. Ki-67-positive cells in the colonies were counted and shown as the Ki-67 labeling index. Scale bars represent 100 µm. E Schematic showing the experimental design for OS tail vein injection using anti-IL-8 antibody. F IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with co-culture CM with or without IL-8 antibodies. G Hematoxylin and eosin staining of lung sections with co-culture CM with or without IL-8 Abs and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. H Immunohistochemistry of Ki-67-positive cells in lung colonies with co-culture CM with or without IL-8 Abs and quantification of Ki-67 labeling index. Scale bars represent 100 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, IVIS, in vivo imaging system, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Injection, Co-Culture Assay, Imaging, Staining, Immunohistochemistry, Labeling, Modification, In Vivo Imaging, Standard Deviation