biotinylated polyclonal rabbit anti-human cxcl8 antibodies  (PeproTech)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Flow chart representing the recruitment of LTx patients for analysis of the neutrophil phenotype (cohort 1) and CXCL8 proteoforms (cohort 2). ( A ) To study the neutrophil phenotype, a first cohort of 100 fresh BAL fluid and 50 peripheral blood samples were collected from 134 individual patients, including 16 patients with a paired BAL fluid and peripheral blood sample. BAL fluid samples were excluded for flow cytometry analysis if cell counts were insufficient or upon severe blood contamination. Samples with insufficient neutrophils for proper flow cytometry analysis were excluded as well. Other reasons for exclusion were samples derived from patients on post-operative day 1 or upon hospital discharge immediately after transplantation, from patients with other severe complications (anastomotic stricture, pulmonary embolisms) or without clear diagnosis. Samples were categorized retrospectively to be derived from LTx patients with CLAD or pulmonary infection or stable LTx recipients according to clinical guidelines (see section). ( B ) To study CXCL8 proteoforms in LTx, COVID-19 and influenza patients, -80 °C-stored BAL fluid supernatants from a second cohort of 55 individual patients were used

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Flow Cytometry, Derivative Assay, Transplantation Assay, Biomarker Discovery, Infection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Clinical characteristics CXCL8 proteoform cohort

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Biomarker Discovery, Cell Counting

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Detection of endogenous CXCL8 proteoforms in BAL fluid. ( A - C ) Neutrophil counts and CXCL8 levels in BAL fluid samples from CLAD ( n = 12), infected ( n = 6) and stable LTx patients ( n = 6) used for CXCL8 proteoform characterization. ( D , E ) Endogenous CXCL8 proteoforms were determined by ISTAMPA in the BAL fluid supernatants of LTx patients with CLAD or infection. Total CXCL8 levels were below the detection limit for ISTAMPA analysis in stable LTx recipients. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( F , G ) Correlation between the relative abundance of the most potent proteoform CXCL8(9–77) and the percentage and absolute numbers of neutrophils in the LTx BAL fluid samples (CLAD and infection combined). ( H , I ) ISTAMPA analysis of endogenous CXCL8 proteoforms in COVID-19 ( n = 24) and influenza ( n = 7) BAL fluid supernatants. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; ** p < 0.01; *** p < 0.001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Proteolytic processing of intact CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant from CLAD ( n = 12), infected ( n = 6) or stable LTx patients ( n = 6) and from patients with COVID-19 ( n = 10) or influenza ( n = 7). After incubation for 3 h at 37 °C, CXCL8 proteolysis was analyzed by ISTAMPA. ( B ) CXCL8 proteoforms were also determined immediately after spiking CXCL8 [without 37 °C incubation but including the required 30 min pre-purification of the CXCL8 proteoforms at room temperature (RT) as part of the ISTAMPA procedure] and after 16 h of incubation at 37 °C in the COVID-19 BAL fluids. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( C , D ) Correlation between the relative abundance of the intact CXCL8(1–77) or the truncated CXCL8(6–77) proteoform after 3 h incubation at 37 °C and the elastinolytic activity in the COVID-19 BAL fluid samples . Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; **** p < 0.0001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Recombinant, Infection, Incubation, Purification, Activity Assay, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Inhibition of proteolytic processing of CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(6–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 3–6) from COVID-19 (indicated by dots) and CLAD patients (indicated by triangles) and incubated for 3 h at 37 °C, in the absence or presence of the metalloprotease inhibitor EDTA. ( B ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 8–9) from COVID-19 (dots) and CLAD patients (triangles) and incubated for 3 h at 37 °C, in the absence or presence of the serine protease inhibitor AEBSF or/and the metalloprotease inhibitor EDTA. After incubation, CXCL8 proteolysis was determined by ISTAMPA. All CXCL8 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL8. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Mann-Whitney U tests or Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; *** p < 0.001; **** p < 0.0001

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Inhibition, Recombinant, Incubation, Protease Inhibitor, Whisker Assay, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Flow chart representing the recruitment of LTx patients for analysis of the neutrophil phenotype (cohort 1) and CXCL8 proteoforms (cohort 2). ( A ) To study the neutrophil phenotype, a first cohort of 100 fresh BAL fluid and 50 peripheral blood samples were collected from 134 individual patients, including 16 patients with a paired BAL fluid and peripheral blood sample. BAL fluid samples were excluded for flow cytometry analysis if cell counts were insufficient or upon severe blood contamination. Samples with insufficient neutrophils for proper flow cytometry analysis were excluded as well. Other reasons for exclusion were samples derived from patients on post-operative day 1 or upon hospital discharge immediately after transplantation, from patients with other severe complications (anastomotic stricture, pulmonary embolisms) or without clear diagnosis. Samples were categorized retrospectively to be derived from LTx patients with CLAD or pulmonary infection or stable LTx recipients according to clinical guidelines (see section). ( B ) To study CXCL8 proteoforms in LTx, COVID-19 and influenza patients, -80 °C-stored BAL fluid supernatants from a second cohort of 55 individual patients were used

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Flow Cytometry, Derivative Assay, Transplantation Assay, Biomarker Discovery, Infection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Clinical characteristics CXCL8 proteoform cohort

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Biomarker Discovery, Cell Counting

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Detection of endogenous CXCL8 proteoforms in BAL fluid. ( A - C ) Neutrophil counts and CXCL8 levels in BAL fluid samples from CLAD ( n = 12), infected ( n = 6) and stable LTx patients ( n = 6) used for CXCL8 proteoform characterization. ( D , E ) Endogenous CXCL8 proteoforms were determined by ISTAMPA in the BAL fluid supernatants of LTx patients with CLAD or infection. Total CXCL8 levels were below the detection limit for ISTAMPA analysis in stable LTx recipients. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( F , G ) Correlation between the relative abundance of the most potent proteoform CXCL8(9–77) and the percentage and absolute numbers of neutrophils in the LTx BAL fluid samples (CLAD and infection combined). ( H , I ) ISTAMPA analysis of endogenous CXCL8 proteoforms in COVID-19 ( n = 24) and influenza ( n = 7) BAL fluid supernatants. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; ** p < 0.01; *** p < 0.001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Infection, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Proteolytic processing of intact CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant from CLAD ( n = 12), infected ( n = 6) or stable LTx patients ( n = 6) and from patients with COVID-19 ( n = 10) or influenza ( n = 7). After incubation for 3 h at 37 °C, CXCL8 proteolysis was analyzed by ISTAMPA. ( B ) CXCL8 proteoforms were also determined immediately after spiking CXCL8 [without 37 °C incubation but including the required 30 min pre-purification of the CXCL8 proteoforms at room temperature (RT) as part of the ISTAMPA procedure] and after 16 h of incubation at 37 °C in the COVID-19 BAL fluids. CXCL8 proteoforms were presented according to their relative abundance in the BAL fluids, expressed as percentage of total CXCL8. ( C , D ) Correlation between the relative abundance of the intact CXCL8(1–77) or the truncated CXCL8(6–77) proteoform after 3 h incubation at 37 °C and the elastinolytic activity in the COVID-19 BAL fluid samples . Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; **** p < 0.0001. Correlation analysis was performed calculating a Spearman correlation coefficient and plotted utilizing a simple linear regression line

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Recombinant, Infection, Incubation, Purification, Activity Assay, Whisker Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Heterogeneous neutrophils in lung transplantation and proteolytic CXCL8 activation in COVID-19, influenza and lung transplant patient lungs

doi: 10.1007/s00018-024-05500-z

Figure Lengend Snippet: Inhibition of proteolytic processing of CXCL8 in BAL fluid. ( A ) Recombinant human CXCL8(6–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 3–6) from COVID-19 (indicated by dots) and CLAD patients (indicated by triangles) and incubated for 3 h at 37 °C, in the absence or presence of the metalloprotease inhibitor EDTA. ( B ) Recombinant human CXCL8(1–77) [125 ng] was spiked in 10 µL of BAL fluid supernatant ( n = 8–9) from COVID-19 (dots) and CLAD patients (triangles) and incubated for 3 h at 37 °C, in the absence or presence of the serine protease inhibitor AEBSF or/and the metalloprotease inhibitor EDTA. After incubation, CXCL8 proteolysis was determined by ISTAMPA. All CXCL8 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL8. Data are shown as box-and-whisker plots (box: median with interquartile range, whiskers: full data distribution), with each dot representing an individual BAL fluid sample, and statistically analyzed by Mann-Whitney U tests or Kruskal-Wallis tests with Dunn’s multiple comparisons tests: * p < 0.05; *** p < 0.001; **** p < 0.0001

Article Snippet: Total CXCL8 concentrations were determined using a specific sandwich ELISA: 0.5 μg/mL polyclonal rabbit anti-human CXCL8 (#500-P28; PeproTech, Cranbury, NJ, USA) and 0.2 μg/mL biotinylated polyclonal rabbit anti-human CXCL8 antibodies (#500-P28BT; PeproTech) with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) were used for coating and detection, respectively.

Techniques: Inhibition, Recombinant, Incubation, Protease Inhibitor, Whisker Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Workflow for immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA). (1) Synovial fluids are collected from the inflamed joints of patients with arthritis. (2) Total CXCL8 is extracted from patient samples by immunosorbent purification using antibody-coupled magnetic beads. (3) Isolated CXCL8 proteoforms are subjected to analysis by nano-UPLC-MS/MS. (4, 5) The detection and quantification of signature fragment ions, generated by top-down collision-induced dissociation (CID) of a pre-selected precursor ion, at the expected elution time point, strongly indicates the presence of a specific CXCL8 proteoform. In general, truncated CXCL8 proteoforms display an enhanced capacity to induce neutrophil migration and activation. This figure is created with BioRender software.

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Purification, Magnetic Beads, Isolation, Tandem Mass Spectroscopy, Generated, Migration, Activation Assay, Software

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms by top-down tandem mass spectrometry. (A) Parameters are optimized to ensure that only the acid-labile Asp-Pro (DP) bond breaks during low-energy fragmentation and only two fragment ions are generated. (B) Mass spectra (single MS) showing the intensity of multiple charged ions of CXCL8(1-77), CXCL8(6-77) and CXCL8(9-77) as a function of their m/z values. Ions with m/z values of 892.8 [for CXCL8(1-77)], 932.3 [for CXCL8(6-77)] or 900.4 [for CXCL8(9-77)] were isolated and fragmented with low energy CID. The resulting fragmentation spectra are shown in the lower panels in (C) . Diamonds indicate the m/z value of the precursor ions selected for fragmentation. Extracted ion chromatograms (EIC) (top panels) show the detection of m/z values (±0.5) of the two signature fragment ions during protein elution. Relative quantification of CXCL8 forms is performed based on the intensity of the two major fragment ions in the EIC. (D) Dose-response curves for the simultaneous quantification of three CXCL8 forms in MRM mode (amounts ranging from 3.1 pg to 12.5 ng). Regression analysis was performed to fit curves to data. Results are represented as mean ± SEM ( n ≥ 4).

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Generated, Isolation, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Site-specific fragmentation of CXCL8 proteoform ions by top-down ion trap tandem mass spectrometry at limited fragmentation energy.

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Sequencing

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms in cell culture supernatant from osteosarcoma cells. The MG-63 osteosarcoma cell line was stimulated with the synthetic double stranded RNA poly rI:rC to produce CXCL8. Partially purified cell culture supernatant was subjected to top-down nano-LC-MS/MS analysis. Two nano-LC-MS/MS runs were performed to examine the presence and quantity of eight CXCL8 proteoforms in MRM mode. (A) Extracted ion chromatograms (EIC) showing the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Fragmentation spectra confirming the presence of signature ions of CXCL8(-2-77), CXCL8(1-77), CXCL8(3-77), CXCL8(6-77), CXCL8(7-77) and CXCL8(8-77). (C) Relative abundance of the detected CXCL8 proteoforms.

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Purification, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 by top down-tandem mass spectrometry proves proteolytic activation in synovial fluids of arthritis patients. Total CXCL8 was extracted from synovial fluids of RA ( n = 14) and JIA patients ( n = 12) by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two top-down nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. A representative experiment is shown (RA patient). (B) Representative fragmentation spectra confirm the presence of signature ions of CXCL8(1-77), CXCL8(6-77), CXCL8(7-77), CXCL8(8-77) and CXCL8(9-77) in synovial fluid from a patient with RA. (C) Relative abundance of CXCL8 proteoforms in synovial fluids from RA and JIA patients (represented as mean ± SEM).

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Mass Spectrometry, Activation Assay, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Kinetics of CXCL8 processing in the presence of synovial fluids from juvenile idiopathic arthritis patients. Exogenous CXCL8(1-77) was incubated with synovial fluids from JIA patients ( n = 4) for a period of 0, 1, 3, 6, 12 or 20 h. Total CXCL8 was extracted from synovial fluids by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Relative abundance of CXCL8 proteoforms in synovial fluids after 0, 1, 3, 6, 12, and 20 h of incubation (represented as mean ± SEM). (C) Pseudo-first order association kinetics of proteolytic modification of CXCL8(1-77) in the presence of synovial fluids (represented as mean ± SEM; n = 4).

Article Snippet: After three washing steps, captured CXCL8 forms were detected using biotinylated polyclonal rabbit anti-human CXCL8 (500-P28BT; PeproTech, Rocky Hill, NJ), raised against recombinant human CXCL8(6-77), combined with peroxidase-conjugated streptavidine (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated, Modification

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Workflow for immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA). (1) Synovial fluids are collected from the inflamed joints of patients with arthritis. (2) Total CXCL8 is extracted from patient samples by immunosorbent purification using antibody-coupled magnetic beads. (3) Isolated CXCL8 proteoforms are subjected to analysis by nano-UPLC-MS/MS. (4, 5) The detection and quantification of signature fragment ions, generated by top-down collision-induced dissociation (CID) of a pre-selected precursor ion, at the expected elution time point, strongly indicates the presence of a specific CXCL8 proteoform. In general, truncated CXCL8 proteoforms display an enhanced capacity to induce neutrophil migration and activation. This figure is created with BioRender software.

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Purification, Magnetic Beads, Isolation, Tandem Mass Spectroscopy, Generated, Migration, Activation Assay, Software

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms by top-down tandem mass spectrometry. (A) Parameters are optimized to ensure that only the acid-labile Asp-Pro (DP) bond breaks during low-energy fragmentation and only two fragment ions are generated. (B) Mass spectra (single MS) showing the intensity of multiple charged ions of CXCL8(1-77), CXCL8(6-77) and CXCL8(9-77) as a function of their m/z values. Ions with m/z values of 892.8 [for CXCL8(1-77)], 932.3 [for CXCL8(6-77)] or 900.4 [for CXCL8(9-77)] were isolated and fragmented with low energy CID. The resulting fragmentation spectra are shown in the lower panels in (C) . Diamonds indicate the m/z value of the precursor ions selected for fragmentation. Extracted ion chromatograms (EIC) (top panels) show the detection of m/z values (±0.5) of the two signature fragment ions during protein elution. Relative quantification of CXCL8 forms is performed based on the intensity of the two major fragment ions in the EIC. (D) Dose-response curves for the simultaneous quantification of three CXCL8 forms in MRM mode (amounts ranging from 3.1 pg to 12.5 ng). Regression analysis was performed to fit curves to data. Results are represented as mean ± SEM ( n ≥ 4).

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Generated, Isolation

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Site-specific fragmentation of CXCL8 proteoform ions by top-down ion trap tandem mass spectrometry at limited fragmentation energy.

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Sequencing

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 proteoforms in cell culture supernatant from osteosarcoma cells. The MG-63 osteosarcoma cell line was stimulated with the synthetic double stranded RNA poly rI:rC to produce CXCL8. Partially purified cell culture supernatant was subjected to top-down nano-LC-MS/MS analysis. Two nano-LC-MS/MS runs were performed to examine the presence and quantity of eight CXCL8 proteoforms in MRM mode. (A) Extracted ion chromatograms (EIC) showing the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Fragmentation spectra confirming the presence of signature ions of CXCL8(-2-77), CXCL8(1-77), CXCL8(3-77), CXCL8(6-77), CXCL8(7-77) and CXCL8(8-77). (C) Relative abundance of the detected CXCL8 proteoforms.

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Cell Culture, Purification, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Detection of CXCL8 by top down-tandem mass spectrometry proves proteolytic activation in synovial fluids of arthritis patients. Total CXCL8 was extracted from synovial fluids of RA ( n = 14) and JIA patients ( n = 12) by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two top-down nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. A representative experiment is shown (RA patient). (B) Representative fragmentation spectra confirm the presence of signature ions of CXCL8(1-77), CXCL8(6-77), CXCL8(7-77), CXCL8(8-77) and CXCL8(9-77) in synovial fluid from a patient with RA. (C) Relative abundance of CXCL8 proteoforms in synovial fluids from RA and JIA patients (represented as mean ± SEM).

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Mass Spectrometry, Activation Assay, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: Frontiers in Immunology

Article Title: From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

doi: 10.3389/fimmu.2021.644725

Figure Lengend Snippet: Kinetics of CXCL8 processing in the presence of synovial fluids from juvenile idiopathic arthritis patients. Exogenous CXCL8(1-77) was incubated with synovial fluids from JIA patients ( n = 4) for a period of 0, 1, 3, 6, 12 or 20 h. Total CXCL8 was extracted from synovial fluids by immunosorbent isolation and subjected to nano-LC-MS/MS analysis. For each sample, two nano-LC-MS/MS runs were performed to examine the presence of eight CXCL8 forms in MRM mode. (A) Extracted ion chromatograms (EIC) show the intensity of m/z values of signature fragment ions (±0.5) generated by fragmentation of a specific precursor ion (indicated between brackets) during protein elution. (B) Relative abundance of CXCL8 proteoforms in synovial fluids after 0, 1, 3, 6, 12, and 20 h of incubation (represented as mean ± SEM). (C) Pseudo-first order association kinetics of proteolytic modification of CXCL8(1-77) in the presence of synovial fluids (represented as mean ± SEM; n = 4).

Article Snippet: Polyclonal rabbit anti-human CXCL8 (60 μg/ml; vide supra ) was immobilized on a carboxyl sensor (Nicoya, Kitchener, ON, Canada) using an OpenSPR benchtop surface plasmon resonance (SPR) instrument (Nicoya).

Techniques: Incubation, Isolation, Liquid Chromatography with Mass Spectroscopy, Generated, Modification

Journal: Scientific Reports

Article Title: Porcine CXCR1/2 antagonist CXCL8 (3–72) G31P inhibits lung inflammation in LPS-challenged mice

doi: 10.1038/s41598-020-57737-w

Figure Lengend Snippet: Recombinant PCXCL8 (3–72) N11R/G31P and PCXCL8 (3–72) G31P protein expression. The gene of porcine CXCL8 (3–72) N11R/G31P and CXCL8 (3–72) G31P were cloned into PGEX 6P-1, and then transformed into E. coli Rosetta (DE3) ( a–c ). Recombinant proteins CXCL8 (3–72) N11R/G31P (pN11R) and CXCL8 (3–72) G31P (pG31P) were induced by IPTG, treated with Prescission Protease, and then purified through GSTrap TM FF. Two proteins molecular weight of 10KD were finally obtained ( d ). The recombinant proteins could be identified by anti-human CXCL8 antibody ( e ).

Article Snippet: The membrane was blocked with 5% skim milk (Sigma, St. Louis, MO, U.S.A.), incubated in mouse anti-human CXCL8 antibody (CSB-PA08327A0Rb, CUSABIO, Wuhan, China) and HRP-conjugated goat-anti-rabbit IgG (CUSABIO, Wuhan, China), and visualized by ECL Western Blotting Substrate (Thermo Fisher, Waltham, MA, U.S.A.).

Techniques: Recombinant, Expressing, Clone Assay, Transformation Assay, Purification, Molecular Weight

Journal: Scientific Reports

Article Title: Porcine CXCR1/2 antagonist CXCL8 (3–72) G31P inhibits lung inflammation in LPS-challenged mice

doi: 10.1038/s41598-020-57737-w

Figure Lengend Snippet: pN11R and pG31P reduce inflammatory factors expression. LPS-challenged mice (LPS + saline) appeared increasing TNF-α, IL-1β and CXCL8 expression compared to saline-challenged mice. The expressions of TNF-α, IL-1β and CXCL8 were also enhanced after treated with pCXCL8 (LPS + pCXCL8), inversely dramatically reduced after treated with pN11R (LPS + pN11R) and pG31P (LPS + pG31P). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t-test, two-tailed.

Article Snippet: The membrane was blocked with 5% skim milk (Sigma, St. Louis, MO, U.S.A.), incubated in mouse anti-human CXCL8 antibody (CSB-PA08327A0Rb, CUSABIO, Wuhan, China) and HRP-conjugated goat-anti-rabbit IgG (CUSABIO, Wuhan, China), and visualized by ECL Western Blotting Substrate (Thermo Fisher, Waltham, MA, U.S.A.).

Techniques: Expressing, Saline, Two Tailed Test

Journal: Scientific Reports

Article Title: Porcine CXCR1/2 antagonist CXCL8 (3–72) G31P inhibits lung inflammation in LPS-challenged mice

doi: 10.1038/s41598-020-57737-w

Figure Lengend Snippet: Primers used for Real-Time PCR.

Article Snippet: The membrane was blocked with 5% skim milk (Sigma, St. Louis, MO, U.S.A.), incubated in mouse anti-human CXCL8 antibody (CSB-PA08327A0Rb, CUSABIO, Wuhan, China) and HRP-conjugated goat-anti-rabbit IgG (CUSABIO, Wuhan, China), and visualized by ECL Western Blotting Substrate (Thermo Fisher, Waltham, MA, U.S.A.).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: The averaged mass spectra of CXCL9(74-93) peptides . The COOH-terminal peptides CXCL9(74-93), NH 2 -terminally biotinylated CXCL9(74-93), and NH 2 -terminally TAMRA-labeled CXCL9(74-93) were chemically synthesized based on Fmoc-chemistry. The intensity of the detected ions in function of their specific mass/charge ( m / z ) ratio is shown for purified CXCL9(74-93) (A) , NH 2 -terminally biotinylated CXCL9(74-93) (B) , and NH 2 -terminally biotinylated TAMRA-labeled CXCL9(74-93) (C) . In order to calculate the relative molecular mass ( M r ) of the proteins corresponding to the indicated ions, Bruker deconvolution software was used. The experimentally determined M r of the peptides are shown as inserts on the upper right of the averaged mass spectra.

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Labeling, Synthesized, Purification, Software

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: CXCL9(74-93) interacts with cellular glycosaminoglycans (GAGs) . The interaction of the COOH-terminal CXCL9 peptides with Chinese hamster ovary (CHO) cells was assessed by flow cytometric analysis. (A) CHO cells were treated with dilutions of NH 2 -terminally biotinylated CXCL9(74-103) and CXCL9(74-93), which were detected by streptavidin-allophycocyanin. (B) To ensure that the binding of CXCL9(74-93) was GAG-mediated, CHO cells were treated with sodium chlorate (NaClO 3 ) to reduce the sulfation of GAGs. Binding of biotinylated CXCL9(74-93) to cells, treated or untreated with NaClO 3 , was detected by streptavidin-allophycocyanin. The mean (+SEM) fluorescence intensity of bound biotinylated CXCL9 peptides to CHO cells is indicated on the y -axis ( n ≥ 4). A statistical comparison to evaluate the binding of biotinylated peptides on CHO cells [compared to the corresponding concentration of CXCL9(74-103) (A) or to the binding to cells not treated with NaClO 3 (B) ] was performed using the Mann–Whitney U -test (* p < 0.05; * * p < 0.01).

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Binding Assay, Fluorescence, Comparison, Concentration Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: CXCL9(74-93) competes with CXCL8 for binding to heparin, heparan sulfate (HS), and cellular glycosaminoglycans (GAGs) . Competition between CXCL8 and COOH-terminal CXCL9 or CXCL4 peptides for binding to immobilized heparin (A) or HS (B) and to Chinese hamster ovary (CHO) cells (C) was performed. (A) and (B) CXCL8(1-77) (0–300 nM) was added to heparin-coated or HS-coated 96-well plates in the presence or absence of the indicated excess of COOH-terminal peptide [CXCL9(74-103) (■), CXCL9(74-93) (□), CXCL9(86-103) (Δ), CXCL4(47-70) (▴)]. Bound CXCL8 was detected with biotinylated anti-human CXCL8 antibodies. (C) Binding of NH 2 -terminally biotinylated CXCL8(1-77) (300 nM) to CHO cells in the presence or absence of an excess CXCL9 peptide was detected by streptavidin-allophycocyanin and flow cytometry. The mean (± SEM) percentage inhibition of binding of CXCL8(1-77) to heparin, HS or cellular GAGs is indicated on the y -axis ( n ≥ 4). Statistical comparison of the different competitors with CXCL9(74–103) was performed using the Mann–Whitney U- test (* p < 0.05).

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Binding Assay, Flow Cytometry, Inhibition, Comparison, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: The COOH-terminal peptides of CXCL9 inhibit CXCL8-induced neutrophil migration to the peritoneal cavity . CXCL8(1-77) was injected into the peritoneal cavity of NMRI mice ( n ≥ 5) simultaneously with an intraperitoneal (A) or intravenous (B,C) injection of CXCL9(74-103) or CXCL9(74–93). The number of neutrophils in the peritoneal cavity, 2 h post injection, were evaluated by differentially counting the leukocyte subtypes on Hemacolor-stained cytospins. The neutrophil counts are shown for every individual mouse, and the horizontal lines denote the median percentage. To detect statistically significant differences, the Mann–Whitney U- test was carried out (* p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Migration, Injection, Staining, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: CXCL9(74-93) inhibits CXCL8-induced but not monosodium urate (MSU) crystal-induced neutrophil migration to the knee cavity . CXCL8(1-77) (1 µg) or MSU crystals (100 µg) were injected into the tibiofemoral articulation of C57BL/6 mice ( n ≥ 5). Simultaneously, 66 µg of CXCL9(74-93) was injected intravenously and 3 h post injection the number of total cells (A) and neutrophils (B) were evaluated by washing the cavity and differentially counting the leukocyte subtypes on May-Grunwald-Giemsa-stained cytospins. The total numbers of leukocytes and neutrophils are shown for every individual mouse, and the horizontal lines denote the median number of cells. To detect statistically significant differences, the Mann–Whitney U -test was carried out (* p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Migration, Injection, Staining, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: CXCL9(74-93) is a less potent competitor for CXCL1 binding to glycosaminoglycans (GAGs) . Competition between CXCL1 and COOH-terminal CXCL9 peptides for binding to immobilized heparan sulfate (HS) (A) and chondroitin sulfate (CS) (B) was performed. Dilutions of CXCL1 were added to GAGs in 96-well plates in the presence or absence of the indicated excess of COOH-terminal peptide [CXCL9(74-103) (■), CXCL9(74-93) (□), CXCL9(86-103) (Δ)]. Bound CXCL1 was detected with biotinylated polyclonal goat anti-mouse CXCL1 antibodies. The mean (± SEM) percentage inhibition of CXCL1 binding to GAGs is indicated on the y- axis ( n ≥ 3). Statistical comparison of the different competitors with CXCL9(74–103) was performed using the Mann–Whitney U- test.

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Binding Assay, Inhibition, Comparison, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: CXCL9(74-103) inhibits adhesion of neutrophils in the murine cremaster muscle model . (A) Neutrophil adhesion to the endothelium in response to an intrascrotal injection of 1 or 3 µg CXCL8 in the presence or absence of 108 µg intravenously injected CXCL9(74-103) was quantified at different time points (45, 60, and 90 min). (B) The cremaster of mice was superfused with 1 (i and iii) or 3 µg (ii and iv) of CXCL8 and endothelium was stained with anti-PECAM-1/CD31 antibody coupled to Alexa 647. The adhering neutrophils were visualized with an anti-Ly6G antibody conjugated to PE and quantified at 60 min post CXCL8 injection. In addition, mice were injected intravenously with either saline (i and ii) or CXCL9(74-103) (iii and iv) 5 min prior to the intrascrotal injection of CXCL8.

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Injection, Staining, Saline

Journal: Frontiers in Immunology

Article Title: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

doi: 10.3389/fimmu.2017.00530

Figure Lengend Snippet: The anti-inflammatory activity of CXCL9(74-93) in murine models of neutrophil migration . In the peritoneum, both CXCL9(74-93) and CXCL9(74-103) inhibit CXCL8-induced neutrophil migration. Because of the high affinity of both peptides for heparan sulfate (HS), it can be suggested that HS is important for the creation of a CXCL8-dependent chemotactic gradient in the peritoneum. In that way, the CXCL9 peptides compete with CXCL8 and inhibit neutrophil extravasation. Although chondroitin sulfate (CS) is most abundant in the articular cavity and the affinity of the peptides for CS is different, both peptides are able to inhibit CXCL8-induced neutrophil migration to the knee cavity. In the gout model, the lower efficiency of CXCL9(74-93) to inhibit CS binding of the induced murine chemokines does not prevent neutrophil migration.

Article Snippet: Subsequently, bound CXCL8 or CXCL1 was detected with biotinylated polyclonal rabbit anti-human CXCL8 or biotinylated polyclonal goat anti-mouse CXCL1 (Peprotech) and horse radish peroxidase-labeled streptavidin.

Techniques: Activity Assay, Migration, Binding Assay