Human IL-8/CXCL8 Biotinylated Antibody

IL-8/CXCL8 in Human PBMCs. IL-8/CXCL8 was detected in immersion fixed PMA-, ionomycin- and monensin-activated human peripheral blood mononuclear cells (PBMCs) using Human IL-8/CXCL8 Biotinylated Antigen Affinity-purified Polyclonal Antibody (Catalog # BAF208) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Streptavidin (red; Catalog # NL999) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Human CXCL8/IL-8 by ELISA Induction of CCL2 and CLXL8 in TNF alpha -stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-alpha (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-alpha. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. beta -Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-alpha (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-alpha in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. #siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results. Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CXCL8/IL-8 by ELISA TNF-alpha upregulates the inflammatory profile of MSCs via receptors TNF-RI and TNF-RII. (A) Relative induction of CCL2, CXCL8 and CCL5 in MSCs by TNF-alpha. Human BM-derived MSCs were stimulated by TNF-alpha (50 ng/ml) for 24 hours and the expression of the inflammatory chemokines CCL2, CXCL8 and CCL5 in supernatants of MSCs was determined by ELISA, in the linear range of absorbance. Results are from an experiment in which all three chemokines were analyzed in parallel, and the ratios between the three chemokines are representatives of the values obtained in many experimental repeats. (B) Human BM-derived MSCs were exposed to neutralizing antibodies against tumor necrosis factor receptors TNF-RI and TNF-RII or nonrelevant isotype-matched control antibodies (Control Ab) 1 hour prior to stimulation with TNF-alpha (50 ng/ml), and in the course of 24 hours of stimulation by the cytokine. Control, cells stimulated with vehicle. Expression of CCL2 was determined by ELISA, in the linear range of absorbance. The findings are representatives of at least n = 3 independent experiments that have shown similar results. Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CXCL8/IL-8 by ELISA TNF-alpha induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-alpha (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-alpha at the last 24 hours of culture (MSCs + TNF-alpha ); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-alpha at the last 24 hours of culture (MSCs + MDA CM + TNF-alpha ). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: *P <0.05, **P ≤0.01, ***P ≤0.001. NS, not significant. #Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant. Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CXCL8/IL-8 by ELISA TNF-alpha induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-alpha (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-alpha at the last 24 hours of culture (MSCs + TNF-alpha ); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-alpha at the last 24 hours of culture (MSCs + MDA CM + TNF-alpha ). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: *P <0.05, **P ≤0.01, ***P ≤0.001. NS, not significant. #Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant. Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CXCL8/IL-8 by ELISA Induction of CCL2 and CLXL8 in TNF alpha -stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-alpha (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-alpha. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. beta -Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-alpha (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-alpha in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. #siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results. Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CXCL8/IL-8 by ELISA Basal and cytokine-induced release of inflammatory chemokines by patient CAFs. CAFs were isolated from lung metastasis of a breast cancer patient (CAFs #1) or from a primary tumor of a different breast cancer patient (CAFs #2). (A1, B1) Expression of the pro-malignancy chemokines CCL2, CXCL8 and CCL5 was determined by ELISA, in the linear range of absorbance. (A2, B2) Expression of CCL5 was determined following TNFa and IL-1 beta stimulation (TNF-alpha, 50 ng/ml; IL-1 beta, 500 pg/ml; 48 hours). Control cells were stimulated by vehicle. Expression of the chemokines was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of at least n = 3 independent experiments that have shown similar results. Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CXCL8/IL-8 by ELISA Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3). Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CXCL8/IL-8 by ELISA Basal and cytokine-induced release of inflammatory chemokines by patient CAFs. CAFs were isolated from lung metastasis of a breast cancer patient (CAFs #1) or from a primary tumor of a different breast cancer patient (CAFs #2). (A1, B1) Expression of the pro-malignancy chemokines CCL2, CXCL8 and CCL5 was determined by ELISA, in the linear range of absorbance. (A2, B2) Expression of CCL5 was determined following TNFa and IL-1 beta stimulation (TNF-alpha, 50 ng/ml; IL-1 beta, 500 pg/ml; 48 hours). Control cells were stimulated by vehicle. Expression of the chemokines was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of at least n = 3 independent experiments that have shown similar results. Image collected and cropped by CiteAb from the following publication (https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0080-7), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human IL-8/CXCL8 Biotinylated Antibody by ELISA Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25928089), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human IL-8/CXCL8 Biotinylated Antibody by ELISA TNF-alpha induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-alpha (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-alpha at the last 24 hours of culture (MSCs + TNF-alpha ); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-alpha at the last 24 hours of culture (MSCs + MDA CM + TNF-alpha ). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: *P <0.05, **P ≤0.01, ***P ≤0.001. NS, not significant. #Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25928089), licensed under a CC-BY license. Not internally tested by R&D Systems.