Journal: International journal of molecular sciences

Article Title: The GAG-Binding Peptide MIG30 Protects against Liver Ischemia-Reperfusion in Mice.

doi: 10.3390/ijms23179715

Figure Lengend Snippet: Figure 5. The MIG30 peptide does not affect the responsiveness or activation status of leukocytes towards stimuli. (A) Intracellular calcium signal ([Ca2+]i in nM) in purified human neutrophils loaded with Fura-2/AM and stimulated with CXCL8 (10 ng/mL) and/or MIG30 (20 µg/mL). Results

Article Snippet: Detection was performed with 250 ng/mL biotinylated polyclonal goat anti-human CXCL8 antibody (BAF208, R&D Systems) for 1 h at 37 ◦C.

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Serum Amyloid A1 (SAA1) Revisited: Restricted Leukocyte-Activating Properties of Homogeneous SAA1

doi: 10.3389/fimmu.2020.00843

Figure Lengend Snippet: hrSAA1 does not induce chemokine expression in CD14 + monocytes and treatment of rSAA1 with LPL diminishes activity. (A,C) Freshly isolated monocytes were induced with homogenous rSAA1 (hrSAA1) (1–12000 ng/ml, 0.08–1000 nM) in parallel with rSAA1 (1–12000 ng/ml, 0.08–1000 nM). (B,D) rSAA1 (100 ng/ml, 8 nM) was pre-incubated with LPL (2000 ng/ml) for 4 h prior to stimulation of monocytes. Following an incubation period of 24 h, cell supernatants were collected and CXCL8 and CCL3 expression was determined via specific ELISAs. Expression in unstimulated cells is indicated by a dashed line (—). Results are represented as the mean chemokine concentration + SEM and are derived from four to six independent experiments. Significant upregulation in comparison to control is indicated by asterisks (Mann-Whitney U -test; * p -value < 0.05, ** p -value < 0.01). Significant reduction in inductive capacity following LPL treatment is indicated by a dagger (Mann-Whitney U -test; † p -value < 0.05).

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and polyclonal goat anti-human CXCL8 (BAF208) antibodies from R&D Systems ( ).

Techniques: Expressing, Activity Assay, Isolation, Incubation, Concentration Assay, Derivative Assay, Comparison, Control, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Serum Amyloid A1 (SAA1) Revisited: Restricted Leukocyte-Activating Properties of Homogeneous SAA1

doi: 10.3389/fimmu.2020.00843

Figure Lengend Snippet: hrSAA1 retains the capacity to synergize with CXCL8 in the in vitro attraction and activation of neutrophils. (A) Homogenous rSAA1 (hrSAA1, 300 or 3000 ng/ml, 25 or 250 nM) and CXCL8 (1–10 ng/ml, 0.12–1.2 nM) were added to the lower compartment of a Boyden microchamber either alone or in combination. Freshly isolated neutrophils were added to the upper compartment and allowed to migrate for 45 min. The chemotactic potencies are expressed as mean chemotactic index (+SEM) derived from six independent experiments. Control recruitment is indicated by a dashed line (—). (B) MMK-1 (16 μg/ml, 9930 nM), hrSAA1 (3000 ng/ml, 250 nM), CXCL8 (3 or 9 ng/ml, 0.36 or 1.07 nM) or a combination of hrSAA1 and CXCL8 were added to the lower compartment of a Boyden microchamber. Freshly isolated neutrophils in the presence or absence of the FPR2 antagonist WRW 4 (20 μg/ml, 18110 nM) were added to the upper compartment and allowed to migrate for 45 min. The chemotactic potencies are expressed as mean chemotactic index + SEM and are derived from three independent experiments. Control recruitment is indicated by a dashed line (—). (C) Neutrophils were stimulated (3 min) with homogenous hrSAA1 (300 or 3000 ng/ml, 25 or 250 nM), CXCL8 (1 or 3 ng/ml, 0.12 or 0.36 nM) or a combination of hrSAA1 and CXCL8. Data from six independent experiments are expressed as the net percentage of activated cells (blebbed and elongated cells) (+SEM). (D) Freshly isolated neutrophils were stimulated for 30 s with different concentrations of hrSAA1, CXCL8 or a combination of hrSAA1 and CXCL8. Following stimulation, cells were stained with Alexa Fluor 555 Phalloidin. Control actin polymerization is indicated by a dashed line (—). Results are presented as the mean percent florescence intensity relative to buffer-stimulated cells (+SEM) and are derived from six independent experiments. Significant neutrophil recruitment/activation in comparison to control is indicated by asterisks (Mann-Whitney U -test; * p -value < 0.05, ** p -value < 0.01). Significant synergy between hrSAA1 and CXCL8 in neutrophil activation/recruitment is indicated by daggers (Mann-Whitney U -test; † p -value < 0.05, †† p -value < 0.01).

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and polyclonal goat anti-human CXCL8 (BAF208) antibodies from R&D Systems ( ).

Techniques: In Vitro, Activation Assay, Isolation, Derivative Assay, Control, Staining, Comparison, MANN-WHITNEY, Significance Assay

Journal: Frontiers in Immunology

Article Title: Serum Amyloid A1 (SAA1) Revisited: Restricted Leukocyte-Activating Properties of Homogeneous SAA1

doi: 10.3389/fimmu.2020.00843

Figure Lengend Snippet: Comparison of the biological activities of E. coli -expressed SAA1 before (SAA1) and after (hrSAA1) purification to homogeneity using RP-HPLC.

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and polyclonal goat anti-human CXCL8 (BAF208) antibodies from R&D Systems ( ).

Techniques: Comparison, Purification, In Vivo, Migration, Expressing

Journal: Mediators of Inflammation

Article Title: Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

doi: 10.1155/2020/6087109

Figure Lengend Snippet: Impure mu rSAA3 induces chemokines in human CD14 + monocytes. Human CD14 + monocytes derived from buffy coats of healthy individuals were stimulated with LPS (500 ng/ml), hu rSAA1 (100 or 1000 ng/ml), mu rSAA3 (10-1000 ng/ml) or were left untreated (Co). After 24 h, supernatants were collected and levels of CXCL8, CCL3 and CCL2 were determined via specific ELISAs. Data represent the mean production of chemokine ± SEM derived from 4 independent experiments. Statistically significant differences from untreated control cells, determined by the Mann-Whitney U test, are indicated by asterisks ( ∗ p < 0.05).

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and biotinylated polyclonal goat anti-human CXCL8 (BAF208) antibodies purchased from R&D Systems (Minneapolis, MN, USA) [ ].

Techniques: Derivative Assay, Control, MANN-WHITNEY

Journal: Mediators of Inflammation

Article Title: Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

doi: 10.1155/2020/6087109

Figure Lengend Snippet: Impure mu rSAA3 is a potent neutrophil chemoattractant in vitro and in vivo and synergizes with CXCL8 in neutrophil migration. (a and b) The chemotactic activity of hu rSAA1 (1000 or 3000 ng/ml) and mu rSAA3 (30-3000 ng/ml) on human neutrophils was evaluated in the Boyden Microchamber Assay. The lower compartment of the microchamber was filled with chemoattractant alone (a) or with a combination of mu rSAA3 (30-3000 ng/ml) and CXCL8 (0.2-3 ng/ml) to assess synergy (b). The chemotactic potency is expressed as chemotactic index and is shown with (a) or without (b) SEM for 5-6 independent experiments. Statistically significant migration (compared to controls) and statistically significant synergy (compared to the sum of the values when both chemoattractants are tested separately), determined by the Mann-Whitney U test, are indicated by asterisks ( ∗ p < 0.05; ∗∗ p < 0.01) and daggers ( † p < 0.05), respectively. (c) Female NMRI mice were injected i.p. with 100 μ l of mu rSAA3 (1-1000 ng), hu rSAA1 (100 ng) or PBS (Co; 2-8 mice per group). After 2 h, mice were sacrificed and peritoneal lavages were performed. Total cell counts in peritoneal lavages were determined and cytospins were prepared for differential leukocyte counts by 2 individuals independently. Data represent the mean percentage (black bars) or absolute number × 10 4 /ml (hatched bars) of neutrophils ± SEM derived from 2 to 3 independent experiments. Statistically significant recruitment of neutrophils compared to PBS-injected mice, determined by the Mann-Whitney U test, is indicated by asterisks ( ∗∗ p < 0.01).

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and biotinylated polyclonal goat anti-human CXCL8 (BAF208) antibodies purchased from R&D Systems (Minneapolis, MN, USA) [ ].

Techniques: In Vitro, In Vivo, Migration, Activity Assay, MANN-WHITNEY, Injection, Derivative Assay

Journal: Mediators of Inflammation

Article Title: Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

doi: 10.1155/2020/6087109

Figure Lengend Snippet: Low concentrations of LPS induce CXCL8 in CD14 + monocytes. Human CD14 + monocytes derived from buffy coats from healthy donors were stimulated with LPS (0.7-7000 pg/ml) or were left untreated (Co). After 24 h, supernatants were collected and levels of CXCL8 were determined via a specific ELISA. Data represent the mean production of CXCL8 ± SEM derived from 3 to 11 independent experiments. Statistically significant differences from untreated control cells, determined by the Mann-Whitney U test, are indicated by asterisks ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and biotinylated polyclonal goat anti-human CXCL8 (BAF208) antibodies purchased from R&D Systems (Minneapolis, MN, USA) [ ].

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY

Journal: Mediators of Inflammation

Article Title: Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

doi: 10.1155/2020/6087109

Figure Lengend Snippet: In contrast to impure mu rSAA3, RP-HPLC-purified mu rSAA3 (mu rSAA3 pur ) does not induce chemokines in leukocytes. Human CD14 + monocytes derived from buffy coats from healthy individuals (a) or murine peritoneal cells derived from healthy mice (b) were stimulated with LPS (500 ng/ml), mu rSAA3 (10 or 100 ng/ml), mu rSAA3 pur (10 or 100 ng/ml) or were left untreated (Co). After 24 h, supernatants were collected and levels of CXCL8 (a) or CXCL2 (b) were determined via specific ELISAs. Data represent the mean production of chemokine ± SEM derived from 4 (a) or 3 (b) independent experiments. Statistically significant differences from untreated control cells, determined by the Mann-Whitney U test, are indicated by asterisks ( ∗ p < 0.05).

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and biotinylated polyclonal goat anti-human CXCL8 (BAF208) antibodies purchased from R&D Systems (Minneapolis, MN, USA) [ ].

Techniques: Purification, Derivative Assay, Control, MANN-WHITNEY

Journal: Mediators of Inflammation

Article Title: Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

doi: 10.1155/2020/6087109

Figure Lengend Snippet: In contrast to impure mu rSAA3, RP-HPLC-purified mu rSAA3 (mu rSAA3 pur ) does not chemoattract neutrophils in vivo . (a) Female NMRI mice were injected i.p. with 100 μ l of PBS (Co), mu rSAA3 (10 or 100 ng) or RP-HPLC-purified mu rSAA3 pur (10 or 100 ng; 3-5 mice per group). After 2 h, mice were sacrificed and peritoneal lavages were performed. Total cell counts in peritoneal lavages were determined and the percentage of neutrophils (Ly-6G + CD11b + ) was quantified via flow cytometry. (b) Female NMRI mice were injected i.p. with 100 μ l of PBS (Co) or LPS (2 or 10 pg; 4-8 mice per group). After 2 h, mice were sacrificed and peritoneal lavages were performed. Total cell counts in peritoneal lavages were determined, and cytospins were prepared for differential leukocyte counts by 2 individuals independently. (a and b) Data represent the mean percentage (black bars) or absolute number × 10 4 /ml (hatched bars) of neutrophils ± SEM derived from 1 (a) or 1-3 (b) independent experiment(s). Statistically significant recruitment of neutrophils compared to PBS-injected mice, determined by the Mann-Whitney U test, is indicated by asterisks ( ∗ p < 0.05; ∗∗ p < 0.01). (c) Human CD14 + monocytes derived from buffy coats from healthy individuals were stimulated with LPS (500 ng/ml), mu rSAA3 (10 or 100 ng/ml), mu rSAA3 pur (10 or 100 ng/ml) or were left untreated (Co). After 2 h, supernatants were collected and levels of CXCL8 were determined via a specific ELISA. Data represent the mean production of chemokine ± SEM derived from 4 independent experiments. Statistically significant differences from untreated control cells, determined by the Mann-Whitney U test, are indicated by asterisks ( ∗ p < 0.05).

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and biotinylated polyclonal goat anti-human CXCL8 (BAF208) antibodies purchased from R&D Systems (Minneapolis, MN, USA) [ ].

Techniques: Purification, In Vivo, Injection, Flow Cytometry, Derivative Assay, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Control

Journal: Mediators of Inflammation

Article Title: Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

doi: 10.1155/2020/6087109

Figure Lengend Snippet: Mu rSAA3 pur synergizes with CXCL8 in neutrophil activation. Activation of human neutrophils was assessed via shape change assays. Neutrophils were stimulated for a period of 3 min with different concentrations of CXCL8 (1 or 3 ng/ml), mu rSAA3 pur (30-3000 ng/ml) or were left untreated. After fixation of cells, non-activated resting (round) and activated (blebbed and elongated) neutrophils were counted microscopically by 2 individuals independently. (a) Data represent the mean net percentage of activated neutrophils ± SEM derived from 4 independent experiments. Statistically significant activation of neutrophils (compared to controls) and statistically significant synergy (compared to the sum of the values when both agonists are tested separately), determined by the Mann-Whitney U test, are indicated by asterisks ( ∗ p < 0.05) and daggers ( † p < 0.05), respectively. (b) Phase contrast image (20x magnification; scale bar: 20 μ m) of one representative experiment illustrating the morphological change of neutrophils following stimulation with buffer (control), CXCL8 (3 ng/ml), mu rSAA3 pur (3000 ng/ml) or a combination of CXCL8 and mu rSAA3 pur are shown.

Article Snippet: The human CXCL8 ELISA was developed in our laboratory using monoclonal mouse anti-human CXCL8 (MAB208) and biotinylated polyclonal goat anti-human CXCL8 (BAF208) antibodies purchased from R&D Systems (Minneapolis, MN, USA) [ ].

Techniques: Activation Assay, Derivative Assay, MANN-WHITNEY, Control