fraction a9  (Teledyne LABS)

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 is not released during exocytosis. ( A ) Neutrophils (5 × 10 5 ) from healthy controls were left unstimulated or incubated with PAF (1 µM, 5 min) or cytochalasin B (5 µg/mL, 5 min) and subsequently stimulated with fMLF (1 µM) for 10 min at 37 °C. To obtain the cytoplasmic fraction of neutrophils, digitonin (150 µM, 15 min) was used to permeabilize the plasma membrane of neutrophils. As a positive control, neutrophils were permeabilized with Triton X-100 (1%, 10 min). S100A8/A9 concentration was determined by ELISA (n = 4–6, mean + SEM). ( B ) Experiment to determine the optimal concentration of digitonin to isolate the cytoplasmic fraction. Lactate dehydrogenase (LDH) levels and protease levels were measured and are presented as percentage of their total content (n = 1). ( C ) Panel I: Lactoferrin concentration in supernatant of unstimulated, PAF/fMLF-stimulated neutrophils, or the cytoplasmic fraction of neutrophils was determined by ELISA (n = 2, mean + SEM). Panel II: Myeloperoxidase concentration in supernatant of unstimulated or CytoB/fMLF-stimulated neutrophils was determined by ELISA (n = 2, mean + SEM). ( D ) Presence of DNA was measured with Sytox Green Nucleic Acid Stain (n = 3, mean + SEM). ( E ) Representative image of digitonin-treated neutrophils. DNA was visualized with Hoechst (blue, 405). Images were acquired using a Leica SP8 confocal microscope, magnification 630x, scale bar = 10 µm. ( F ) Coomassie Brilliant Blue staining and Western Blot for S100A8 (red) and S100A9 (green) of the cytoplasmic fraction of neutrophils. ( G ) Representative immuno-electron microscopy image of a neutrophil (right) and lymphocyte (left) stained for anti-S100A8 (black dots). Scale bar = 1 µm. “A” indicates azurophilic granules and “S” indicates specific granules. S100A8 is not present in neutrophil granules. Statistics were performed by mixed effects’ model ( A ) or one-way ANOVA ( D) with Dunnett post hoc test; ** p < 0.01, *** p < 0.001, ns = not significant; n.d. = not detectable.

Article Snippet: Neutrophils (1 × 10 5 ) were incubated with tumor necrosis factor (TNF-α; 10 ng/mL Peprotech, London, UK) or the isolated S100A8/A9 fraction for 2 h at 37 °C.

Techniques: Incubation, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Western Blot, Immuno-Electron Microscopy

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 release by neutrophils upon NETosis. To induce NET formation, neutrophils were stimulated with ( A – E ) PMA (100 ng/mL) or ( F – H ) MSU crystals (200 µg/mL) for 4 h at 37 °C. ( A , B , F ) NETs were visualized by staining for Elastase (ELA) (green, 488), DNA (Hoechst, blue, 405), and ( A ) S100A8/A9 or ( B , F ) myeloperoxidase (MPO) (magenta, 633). Images were acquired using a Leica SP8 confocal microscope, magnification 400×. Scale bar = ( A , F ) 100 µm or ( B ) 75 µm. ( C , G ) DNA release of neutrophils was measured by Sytox Green Nucleic Acid Stain upon stimulation with ( C ) PMA (n = 8 for controls and n = 4 unique CGD patients, of which one patient was measured three times, mean + SEM) or ( G ) MSU crystals (n = 5 for controls and n = 3 unique CGD patients, of which one patient was measured two times, mean + SEM). Triton X-100 was used as positive control. ( D , E ) Supernatant of PMA-stimulated neutrophils was collected at the indicated time points and measured for ( D ) S100A8/A9 and ( E ) lactoferrin by ELISA. ( F ) Arrows indicate NET structures around MSU crystals. ( H ) Supernatants of neutrophils stimulated for 4 h with PMA or MSU crystals were collected and measured for S100A8/A9 by ELISA (n = 2 for controls, n = 2 for CGD patients, mean + SEM). CGD = chronic granulomatous disease.

Article Snippet: Neutrophils (1 × 10 5 ) were incubated with tumor necrosis factor (TNF-α; 10 ng/mL Peprotech, London, UK) or the isolated S100A8/A9 fraction for 2 h at 37 °C.

Techniques: Staining, Microscopy, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 purification of the cytoplasm of neutrophils. ( A ) Schematic overview of the purification process of S100A8/A9 from neutrophil cytosol. ( B ) The cytoplasmic fraction of neutrophils was isolated followed by 50% ammonium sulfate (AS) precipitation and anion-exchange chromatography (AEX). Indicated are the different peaks that were isolated (C2–C9). ( C ) Coomassie Brilliant Blue staining and ( D ) Western blot of isolated peak fractions, identifying S100A8 (red) and S100A9 (green) to be presented as mostly heterodimers and monomers. ( E ) Coomassie Brilliant Blue staining with a human serum albumin (HSA) calibration curve that was used to calculate the concentration of S100A8 and A9 heterodimers and monomers in the S100A8/A9 fraction used for functional experiments.

Article Snippet: Neutrophils (1 × 10 5 ) were incubated with tumor necrosis factor (TNF-α; 10 ng/mL Peprotech, London, UK) or the isolated S100A8/A9 fraction for 2 h at 37 °C.

Techniques: Purification, Isolation, Chromatography, Staining, Western Blot, Concentration Assay, Functional Assay

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 activates neutrophils. ( A ) Viability of neutrophils upon 2 h of incubation at 37 °C with S100A8/A9 was assessed by flow cytometry for AnnexinV and TO-PRO. Viable neutrophils were defined as negative for both markers (n = 3, mean + SEM). Indicated in the left panel is the gating strategy. ( B , C ) Expression of neutrophil activation markers ( B ) CD11b (n = 3–5, mean + SEM) and ( C ) CD62L (n = 2, mean + SEM) was measured by flow cytometry upon 2 h of incubation at 37 °C with indicated concentrations of S100A8/A9 or TNF-α in the absence or presence of 10% human serum. ( B ) Indicated in the left panel is the gating strategy. ( C ) FACS plots showing CD62-L shedding are displayed. ( D ) Neutrophils were left unstimulated or stimulated with LPS/LBP or different concentrations of S100A8/A9 for 30 min at 37 °C in the presence of 10% human serum. Percentage of adhesion is shown compared to total input (n = 3–5, mean + SEM). ( E ) Neutrophils were primed by LPS/LBP or different concentrations of S100A8/A9 in the presence of 10% human serum. After 30 min of incubation at 37 °C, 1 µM fMLF was added to induce ROS production. The maximal slope of H 2 O 2 production was measured using Amplex Red (n = 5, mean + SEM). Statistics were performed by paired t-test ( A , B , E ) or mixed effects’ model with Dunnett post hoc test ( D ); * p < 0.05, ** p < 0.01, ns = not significant.

Article Snippet: Neutrophils (1 × 10 5 ) were incubated with tumor necrosis factor (TNF-α; 10 ng/mL Peprotech, London, UK) or the isolated S100A8/A9 fraction for 2 h at 37 °C.

Techniques: Incubation, Flow Cytometry, Expressing, Activation Assay

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 is not released during exocytosis. ( A ) Neutrophils (5 × 10 5 ) from healthy controls were left unstimulated or incubated with PAF (1 µM, 5 min) or cytochalasin B (5 µg/mL, 5 min) and subsequently stimulated with fMLF (1 µM) for 10 min at 37 °C. To obtain the cytoplasmic fraction of neutrophils, digitonin (150 µM, 15 min) was used to permeabilize the plasma membrane of neutrophils. As a positive control, neutrophils were permeabilized with Triton X-100 (1%, 10 min). S100A8/A9 concentration was determined by ELISA (n = 4–6, mean + SEM). ( B ) Experiment to determine the optimal concentration of digitonin to isolate the cytoplasmic fraction. Lactate dehydrogenase (LDH) levels and protease levels were measured and are presented as percentage of their total content (n = 1). ( C ) Panel I: Lactoferrin concentration in supernatant of unstimulated, PAF/fMLF-stimulated neutrophils, or the cytoplasmic fraction of neutrophils was determined by ELISA (n = 2, mean + SEM). Panel II: Myeloperoxidase concentration in supernatant of unstimulated or CytoB/fMLF-stimulated neutrophils was determined by ELISA (n = 2, mean + SEM). ( D ) Presence of DNA was measured with Sytox Green Nucleic Acid Stain (n = 3, mean + SEM). ( E ) Representative image of digitonin-treated neutrophils. DNA was visualized with Hoechst (blue, 405). Images were acquired using a Leica SP8 confocal microscope, magnification 630x, scale bar = 10 µm. ( F ) Coomassie Brilliant Blue staining and Western Blot for S100A8 (red) and S100A9 (green) of the cytoplasmic fraction of neutrophils. ( G ) Representative immuno-electron microscopy image of a neutrophil (right) and lymphocyte (left) stained for anti-S100A8 (black dots). Scale bar = 1 µm. “A” indicates azurophilic granules and “S” indicates specific granules. S100A8 is not present in neutrophil granules. Statistics were performed by mixed effects’ model ( A ) or one-way ANOVA ( D) with Dunnett post hoc test; ** p < 0.01, *** p < 0.001, ns = not significant; n.d. = not detectable.

Article Snippet: In brief, for adhesion experiments, neutrophils were labeled with calcein-AM (1 μM; Molecular Probes, Eugene, OR, USA) and stimulated with either lipopolysaccharide (LPS) and LBP (LPS-binding protein), the S100A8/A9 fraction, or left unstimulated and incubated for 30 min at 37 °C in an uncoated 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany).

Techniques: Incubation, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Western Blot, Immuno-Electron Microscopy

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 release by neutrophils upon NETosis. To induce NET formation, neutrophils were stimulated with ( A – E ) PMA (100 ng/mL) or ( F – H ) MSU crystals (200 µg/mL) for 4 h at 37 °C. ( A , B , F ) NETs were visualized by staining for Elastase (ELA) (green, 488), DNA (Hoechst, blue, 405), and ( A ) S100A8/A9 or ( B , F ) myeloperoxidase (MPO) (magenta, 633). Images were acquired using a Leica SP8 confocal microscope, magnification 400×. Scale bar = ( A , F ) 100 µm or ( B ) 75 µm. ( C , G ) DNA release of neutrophils was measured by Sytox Green Nucleic Acid Stain upon stimulation with ( C ) PMA (n = 8 for controls and n = 4 unique CGD patients, of which one patient was measured three times, mean + SEM) or ( G ) MSU crystals (n = 5 for controls and n = 3 unique CGD patients, of which one patient was measured two times, mean + SEM). Triton X-100 was used as positive control. ( D , E ) Supernatant of PMA-stimulated neutrophils was collected at the indicated time points and measured for ( D ) S100A8/A9 and ( E ) lactoferrin by ELISA. ( F ) Arrows indicate NET structures around MSU crystals. ( H ) Supernatants of neutrophils stimulated for 4 h with PMA or MSU crystals were collected and measured for S100A8/A9 by ELISA (n = 2 for controls, n = 2 for CGD patients, mean + SEM). CGD = chronic granulomatous disease.

Article Snippet: In brief, for adhesion experiments, neutrophils were labeled with calcein-AM (1 μM; Molecular Probes, Eugene, OR, USA) and stimulated with either lipopolysaccharide (LPS) and LBP (LPS-binding protein), the S100A8/A9 fraction, or left unstimulated and incubated for 30 min at 37 °C in an uncoated 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany).

Techniques: Staining, Microscopy, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 purification of the cytoplasm of neutrophils. ( A ) Schematic overview of the purification process of S100A8/A9 from neutrophil cytosol. ( B ) The cytoplasmic fraction of neutrophils was isolated followed by 50% ammonium sulfate (AS) precipitation and anion-exchange chromatography (AEX). Indicated are the different peaks that were isolated (C2–C9). ( C ) Coomassie Brilliant Blue staining and ( D ) Western blot of isolated peak fractions, identifying S100A8 (red) and S100A9 (green) to be presented as mostly heterodimers and monomers. ( E ) Coomassie Brilliant Blue staining with a human serum albumin (HSA) calibration curve that was used to calculate the concentration of S100A8 and A9 heterodimers and monomers in the S100A8/A9 fraction used for functional experiments.

Article Snippet: In brief, for adhesion experiments, neutrophils were labeled with calcein-AM (1 μM; Molecular Probes, Eugene, OR, USA) and stimulated with either lipopolysaccharide (LPS) and LBP (LPS-binding protein), the S100A8/A9 fraction, or left unstimulated and incubated for 30 min at 37 °C in an uncoated 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany).

Techniques: Purification, Isolation, Chromatography, Staining, Western Blot, Concentration Assay, Functional Assay

Journal: Cells

Article Title: S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

doi: 10.3390/cells11020236

Figure Lengend Snippet: S100A8/A9 activates neutrophils. ( A ) Viability of neutrophils upon 2 h of incubation at 37 °C with S100A8/A9 was assessed by flow cytometry for AnnexinV and TO-PRO. Viable neutrophils were defined as negative for both markers (n = 3, mean + SEM). Indicated in the left panel is the gating strategy. ( B , C ) Expression of neutrophil activation markers ( B ) CD11b (n = 3–5, mean + SEM) and ( C ) CD62L (n = 2, mean + SEM) was measured by flow cytometry upon 2 h of incubation at 37 °C with indicated concentrations of S100A8/A9 or TNF-α in the absence or presence of 10% human serum. ( B ) Indicated in the left panel is the gating strategy. ( C ) FACS plots showing CD62-L shedding are displayed. ( D ) Neutrophils were left unstimulated or stimulated with LPS/LBP or different concentrations of S100A8/A9 for 30 min at 37 °C in the presence of 10% human serum. Percentage of adhesion is shown compared to total input (n = 3–5, mean + SEM). ( E ) Neutrophils were primed by LPS/LBP or different concentrations of S100A8/A9 in the presence of 10% human serum. After 30 min of incubation at 37 °C, 1 µM fMLF was added to induce ROS production. The maximal slope of H 2 O 2 production was measured using Amplex Red (n = 5, mean + SEM). Statistics were performed by paired t-test ( A , B , E ) or mixed effects’ model with Dunnett post hoc test ( D ); * p < 0.05, ** p < 0.01, ns = not significant.

Article Snippet: In brief, for adhesion experiments, neutrophils were labeled with calcein-AM (1 μM; Molecular Probes, Eugene, OR, USA) and stimulated with either lipopolysaccharide (LPS) and LBP (LPS-binding protein), the S100A8/A9 fraction, or left unstimulated and incubated for 30 min at 37 °C in an uncoated 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany).

Techniques: Incubation, Flow Cytometry, Expressing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: miRNAs Regulate Cytokine Secretion Induced by Phosphorylated S100A8/A9 in Neutrophils

doi: 10.3390/ijms20225699

Figure Lengend Snippet: Presence of S100A8/A9-P in synovial fluids from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. 50 µg of proteins from synovial fluids from 14 RA and 14 OA patients were loaded on a 4–15% Tris-Acetate gel for western blot analysis. S100A9-P was detected with the anti-S100A9-P antibody (custom-made), total S100A9 with the B5 antibody and S100A8 using the EPR3554 antibody. (L = ladder and positive control = leukopak purified S100A8/A9-P).

Article Snippet: Finally, S100A8/A9 or S100A8/A9-P fractions were pooled and concentrated to a workable concentration using Amicon ® Ultra 3K Centrifugal Filter Units (Merck, Darmstadt, Germany).

Techniques: Western Blot, Positive Control, Purification

Journal: International Journal of Molecular Sciences

Article Title: miRNAs Regulate Cytokine Secretion Induced by Phosphorylated S100A8/A9 in Neutrophils

doi: 10.3390/ijms20225699

Figure Lengend Snippet: Effect of S100A8/A9-P on miRNA expression in dHL-60 cells. dHL-60 cells were stimulated for 3, 6 and 12 h with 10 µg/mL S100A8/A9-P. ( A ) Volcano plot represents the log2 FC (fold change) (x-axis) against the -log10 p-values (y-axis) for each miRNA identified using miRNA-sequencing. ( B ) MA plot represents the average log CPM (count per million) value (x-axis) against the log2 FC (y-axis) for each miRNA identified using miRNA-sequencing. From three independent experiments, criteria for an effect of S100A8/A9-P on miRNA expression were defined as follows: FDR < 5%, log2 FC ≥ 0.5 and average trimmed mean of M (TMM) value difference between non-stimulated dHL-60 cells and S100A8/A9-P stimulated dHL-60 cells ≥ 100. ( C ) dHL-60 cells were stimulated for 3, 6, 12, 18 and 24 h with 10 µg/mL S100A8/A9-P or S100A8/A9. The expression of miR-146a-5p and miR-155-5p was assessed using qPCR. Data normalization was performed using three reference genes (RNUA1, RNUA5, SCARNA17) and expressed as fold induction compared to the non-stimulated control (dashed line). Results are presented as mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Finally, S100A8/A9 or S100A8/A9-P fractions were pooled and concentrated to a workable concentration using Amicon ® Ultra 3K Centrifugal Filter Units (Merck, Darmstadt, Germany).

Techniques: Expressing, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: miRNAs Regulate Cytokine Secretion Induced by Phosphorylated S100A8/A9 in Neutrophils

doi: 10.3390/ijms20225699

Figure Lengend Snippet: S100A8/A9-P-induced miR-146a and miR-155-5p expression through TLR4 signaling pathway in dHL-60 cells. dHL-60 cells were incubated for 30 min with TLR4 neutralizing antibody, then stimulated with 10 µg/mL S100A8/A9-P for 18 h. 1 µg/mL TLR4 neutralizing antibody was added at 6 h and 12 h of stimulation. Expression of ( A ) miR-146a-5p and ( B ) miR-155-5p were quantified using RT-qPCR. Data normalization was performed using three reference genes (RNUA1, RNUA5, SCARNA17) and expressed as fold induction compared to the non-stimulated control. Results are presented as mean ± SEM of three independent experiments. ** p < 0.01; **** p < 0.0001.

Article Snippet: Finally, S100A8/A9 or S100A8/A9-P fractions were pooled and concentrated to a workable concentration using Amicon ® Ultra 3K Centrifugal Filter Units (Merck, Darmstadt, Germany).

Techniques: Expressing, Incubation, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: miRNAs Regulate Cytokine Secretion Induced by Phosphorylated S100A8/A9 in Neutrophils

doi: 10.3390/ijms20225699

Figure Lengend Snippet: Effect of miR-146a-5p and miR-155-5p mimics on cytokine secretion induced by S100A8/A9-P in dHL-60 cells. Transduced dHL-60 cells with miR-146a-5p and miR-155-5p were stimulated with 10 µg/mL S100A8/A9-P for 6 h. Secretion of CCL2 and CXCL8 was quantified using cytometric bead array and secretion of CCL4 by ELISA. Cell viability for each condition was measured using LDH quantification. The secretion results are presented as mean ± SEM of six independent experiments. *** p < 0.001; **** p < 0.0001.

Article Snippet: Finally, S100A8/A9 or S100A8/A9-P fractions were pooled and concentrated to a workable concentration using Amicon ® Ultra 3K Centrifugal Filter Units (Merck, Darmstadt, Germany).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: S100A9 Induced Inflammatory Responses Are Mediated by Distinct Damage Associated Molecular Patterns (DAMP) Receptors In Vitro and In Vivo

doi: 10.1371/journal.pone.0115828

Figure Lengend Snippet: Human PBMCs were stimulated with 10 μg/ml of human S100 preparations and levels of IL-6 (and other cytokines) were measured after 16 h in culture. (A) Cells were stimulated with low endotoxin preparations of calgranulins (S100A8, S100A9 or S100A12) or LPS and were treated with either nil, control Ab, anti-hTLR4, anti-RAGE Abs or Polymyxin B (PMB). (B) Cells were stimulated with S100A1, S100A16, S100B and S100A8/A9 and were similarly treated with nil, control Ab, anti-hTLR4 Ab, anti-RAGE Ab, or Polymyxin B. Representative data from one of three experiments is shown. Individual experiments were performed in triplicate and mean ± SD are given. Unpaired T-tests was used to determine if the test antibody differed significantly from their respective control Abs following stimulation, and if the polymyxin treatment differed from no treatment following stimulation (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: Pooled S100A8/A9 fractions were diluted 10-fold with Buffer C (50 mM sodium acetate (pH 4.5), 1 mM EDTA, 1 mM DTT, 1 mM CaCl 2 ) and then positive fractions were applied to a HiTrap SP-HP column (GE Healthcare, Pittsburgh, PA).

Techniques:

Journal: PLoS ONE

Article Title: S100A9 Induced Inflammatory Responses Are Mediated by Distinct Damage Associated Molecular Patterns (DAMP) Receptors In Vitro and In Vivo

doi: 10.1371/journal.pone.0115828

Figure Lengend Snippet: Migration indices for THP-1 cells in response to hS100A8 (A), hS100A9 (B), and hS100A12 (C). Effects of anti-RAGE, anti-hTLR4 Abs and isotype control Abs on hS100A8 (D), hS100A9 (E) and hS100A12 (F) induced THP-1 cell migration. Serial dilution of anti-RAGE, anti-TLR4 or isotype-matched control Ab were incubated with THP-1 cells in the upper wells of the chemotaxis chamber, and optimal amounts of hS100A8 (1 ng/ml), hS100A9 (1 ng/ml) and hS100A12 (100 ng/ml) were added in the lower wells. Percentage inhibition is relative to no Ab treatment. One representative of three independent experiments is shown (mean ±SD of triplicate wells) (G) The effects of a fixed 10 μg/ml dose of anti-RAGE, anti-hTLR4 and control Abs on THP-1 cell migration mediated by other S100s and MCP1. The S100s were used at their optimal concentrations (indicated on graphs). The maximal chemotactic indexes were S100A1 (2.0), S100A4 (2.9), S100A6 (2.9), S100A7 (3.7), S100A8/A9 (3.1), S100A10 (1.9), S100A14 (2.7), S100A16 (2.4), S100P (3.5), S100B (3.5) and MCP1 (9.5). Percentage inhibition is relative to no Ab treatment. Mean inhibition ± SD for one of two independent experiments is shown. Unpaired T-test was used to determine if the anti-RAGE or anti-hTLR4 Ab treatments were significantly different from the isotype control Ab (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: Pooled S100A8/A9 fractions were diluted 10-fold with Buffer C (50 mM sodium acetate (pH 4.5), 1 mM EDTA, 1 mM DTT, 1 mM CaCl 2 ) and then positive fractions were applied to a HiTrap SP-HP column (GE Healthcare, Pittsburgh, PA).

Techniques: Migration, Serial Dilution, Incubation, Chemotaxis Assay, Inhibition