Journal: Biomolecules and Biomedicine

Article Title: The link between neutrophils, NETs, and NLRP3 inflammasomes: The dual effect of CD177 and its therapeutic potential in acute respiratory distress syndrome/acute lung injury

doi: 10.17305/bb.2023.10101

Figure Lengend Snippet: Flow cytometry gating strategy. (A) We delineated live cells, excluded dead cells and cell debris; (B) and then removed the adhesions; (C) The fluorescence thresholds of FITC and (D) PE were set using Isotype Control antibodies IgG; (E) After FITC-CD66b + /Ly-6G + neutrophils were isolated by flow sorting, PE-CD177 + fluorescent antibody was used to (F) further detect the CD177 + neutrophils ratio.

Article Snippet: Following blocking with goat serum, the slides were incubated overnight in a humidified chamber at 4 ∘ C with a mouse anti-human CD177 antibody (1:200; Abcam, UK).

Techniques: Flow Cytometry, Fluorescence, Control, Isolation

Journal: Biomolecules and Biomedicine

Article Title: The link between neutrophils, NETs, and NLRP3 inflammasomes: The dual effect of CD177 and its therapeutic potential in acute respiratory distress syndrome/acute lung injury

doi: 10.17305/bb.2023.10101

Figure Lengend Snippet: CD177 expression in ARDS patients. (A) Flow cytometry analysis illustrating the percentage of CD177+ neutrophils in peripheral blood; (B) Neutrophils were isolated from the peripheral blood of healthy volunteers (Control, n ═ 6) and ARDS patients (ARDS, n ═ 6). Flow cytometry was employed to detect CD66b+ neutrophils and CD177+ CD66b+ neutrophils; (C) The levels of common inflammatory factors in the peripheral blood of healthy controls and ARDS patients were measured using ELISA kits, following the manufacturer’s instructions. * P < 0.05, ** P < 0.01, *** P < 0.001. ARDS: Acute respiratory distress syndrome; DEG: Differentially expressed gene; ELISA: Enzyme-linked immunosorbent assay.

Article Snippet: Following blocking with goat serum, the slides were incubated overnight in a humidified chamber at 4 ∘ C with a mouse anti-human CD177 antibody (1:200; Abcam, UK).

Techniques: Expressing, Flow Cytometry, Isolation, Control, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules and Biomedicine

Article Title: The link between neutrophils, NETs, and NLRP3 inflammasomes: The dual effect of CD177 and its therapeutic potential in acute respiratory distress syndrome/acute lung injury

doi: 10.17305/bb.2023.10101

Figure Lengend Snippet: CD177 inhibition can attenuate the acute inflammatory response of LPS-induced ALI . We established a mouse model of lung injury with LPS (A) and treated it with anti-CD177 antibody (0.1 ng) by intraperitoneal injection, with IgG antibody as a control (0.1 ng). After 48 h, we observed a significant reduction in pulmonary inflammation (B), along with a lower percentage of CD177+ neutrophils in peripheral blood (C and D), improved survival rate (E), and clinical symptom score (F) in mice. Lung tissue sections also showed the difference between the LPS and anti-CD177 antibody treatment groups (G). * P < 0.05, ** P < 0.01, *** P < 0.001. LPS: Lipopolysaccharide; ALI: Acute lung injury.

Article Snippet: Following blocking with goat serum, the slides were incubated overnight in a humidified chamber at 4 ∘ C with a mouse anti-human CD177 antibody (1:200; Abcam, UK).

Techniques: Inhibition, Injection, Control

Journal: Biomolecules and Biomedicine

Article Title: The link between neutrophils, NETs, and NLRP3 inflammasomes: The dual effect of CD177 and its therapeutic potential in acute respiratory distress syndrome/acute lung injury

doi: 10.17305/bb.2023.10101

Figure Lengend Snippet: CD177 plays an important mediating role in LPS-induced ALI. (A) Blocking the CD177 antigen on the neutrophil surface reduces pulmonary edema; (B) diminishes alveolar neutrophil infiltration, lung tissue MPO levels; (C) and ROS levels; (D–G) After 48 h of establishing the mouse ALI model, levels of common inflammatory factors in the BALF and (H–K) PB were measured using ELISA kits, according to the manufacturer’s instructions. * P < 0.05, ** P < 0.01, *** P < 0.001. LPS: Lipopolysaccharide; ALI: Acute lung injury; BALF: Bronchoalveolar lavage fluid; ELISA: Enzyme-linked immunosorbent assay.

Article Snippet: Following blocking with goat serum, the slides were incubated overnight in a humidified chamber at 4 ∘ C with a mouse anti-human CD177 antibody (1:200; Abcam, UK).

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules and Biomedicine

Article Title: The link between neutrophils, NETs, and NLRP3 inflammasomes: The dual effect of CD177 and its therapeutic potential in acute respiratory distress syndrome/acute lung injury

doi: 10.17305/bb.2023.10101

Figure Lengend Snippet: Impact of CD177 on the formation and release of the inflammasomes in neutrophils. (A–D) We measured the levels of inflammasome-related proteins NLRP3 and caspase-1; (E and F) Based on the WB method, we detected the PAD4 expression levels, a marker of NETs; (G and H) We used ELISA kits to measure the concentrations of inflammatory factors in the supernatants; (I and J) We used ROS and MPO kits to measure the relative levels of MPO and ROS in the cells. * P < 0.05, ** P < 0.01, *** P < 0.001. NET: Neutrophil extracellular trap; ELISA: Enzyme-linked immunosorbent assay; ROS: Reactive oxygen species; MPO: Myeloperoxidase.

Article Snippet: Following blocking with goat serum, the slides were incubated overnight in a humidified chamber at 4 ∘ C with a mouse anti-human CD177 antibody (1:200; Abcam, UK).

Techniques: Expressing, Marker, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules and Biomedicine

Article Title: The link between neutrophils, NETs, and NLRP3 inflammasomes: The dual effect of CD177 and its therapeutic potential in acute respiratory distress syndrome/acute lung injury

doi: 10.17305/bb.2023.10101

Figure Lengend Snippet: Mechanistic diagram of CD177 in neutrophils. ROS: Reactive oxygen species; MPO: Myeloperoxidase; LPS: Lipopolysaccharide; DNA: Deoxyribonucleic acid.

Article Snippet: Following blocking with goat serum, the slides were incubated overnight in a humidified chamber at 4 ∘ C with a mouse anti-human CD177 antibody (1:200; Abcam, UK).

Techniques:

Journal: EMBO Reports

Article Title: Succinate induces skeletal muscle fiber remodeling via SUNCR 1 signaling

doi: 10.15252/embr.201947892

Figure Lengend Snippet: Male C57BL/6J mice were fed with chow diet supplemented with 0 and 1% SUC for 6 weeks. A–D The O 2 consumption (VO 2 ) (A, B) and respiratory exchange ratio (RER) (C, D). E–H Serum concentration of (E) NEFA in whole blood. The enzymes activity of (F) SDH, (G) HK, and (H) LDH in gastrocnemius. I, J Immunoblots and quantification of p‐AMPK, PGC‐1α, and myoglobin in gastrocnemius. The same lysates were used for the detection of PGC1α (100 kDa, Fig I), myoglobin (17 kDa, Fig I), myosin heavy chain (180 kDa, Fig B), and tubulin (48 kDa, shared in both Figs B and I). K Quantification of mitochondrial and electron transport chain (ETC)‐related gene expression i respiratory exchange n gastrocnemius. L OCRs were measured under basal condition in gastrocnemius. Data information: Results are presented as mean ± SEM ( n = 4–6). Different letters between bars mean P ≤ 0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests. * P ≤ 0.05 and ** P ≤ 0.01 by non‐paired Student's t ‐test.

Article Snippet: After sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis gels, primary antibodies were used, including rabbit anti‐β‐tubulin (bs‐1482M, 1:5,000, Bioss), rabbit anti‐SUNCR1 (NBP1‐00861, 1:1,000, Novus), mouse anti‐MyHC I (ab11083, 1:1,000; Abcam), rabbit anti‐MyHC IIa (ab124937, 1:1,000, Abcam), goat anti‐MyHC IIb (sc‐168672, 1:500; Santa Cruz), mouse anti‐PGC‐1α (ST1202, 1:1,000, Millipore), rabbit anti‐histone (4499S, 1:2,000; CST), mouse anti‐NFAT (sc‐7294, 1:500; Santa Cruz), rabbit anti‐NRF‐1 (#12381s, 1:2,000, CST), rabbit anti‐calcineurin (#2614s, 1:2,000; CST), rabbit anti‐Myoglobin (ab77232, 1:1,000, Abcam), and rabbit anti‐MEF2A (#97365, 1:2,000; CST).

Techniques: Concentration Assay, Activity Assay, Western Blot, Expressing

Journal: BMC Gastroenterology

Article Title: Gene expression analysis of a Helicobacter pylori -infected and high-salt diet-treated mouse gastric tumor model: identification of CD177 as a novel prognostic factor in patients with gastric cancer

doi: 10.1186/1471-230X-13-122

Figure Lengend Snippet: Primer sequences for relative quantitative real-time RT-PCR

Article Snippet: Then, sections were incubated with a mouse monoclonal anti-CD177 antibody (clone 4C4, diluted 1:100, Abnova, Taipei, Taiwan).

Techniques:

Journal: BMC Gastroenterology

Article Title: Gene expression analysis of a Helicobacter pylori -infected and high-salt diet-treated mouse gastric tumor model: identification of CD177 as a novel prognostic factor in patients with gastric cancer

doi: 10.1186/1471-230X-13-122

Figure Lengend Snippet: Global gene analysis in the glandular stomach of MNU-treated mice using oligonucleotide microarray. A : Number of genes up- or down-regulated more than two-fold in the stomach of MNU-treated mice. In Venn’s diagram, the circles indicate up- (left) or down-regulated (right) genes in the stomach of MNU-treated mice with H. pylori infection, high-salt diet or their combination. The shaded area represents the up- or down-regulated genes more than two-fold only by the combination. B : Quantitative real-time RT-PCR analysis of three selected up-regulated genes ( Cd177 , Reg3g , and Muc13 ) in the stomachs of MNU-treated mice. Expression levels of the genes in each sample were normalized by Gapdh as internal control using ΔΔCT method. Relative expression levels were represented as the X-fold change relative to Group A (fixed as 1.0). Statistical analysis was performed by the Kruskal-Wallis test for general analysis and Tukey test for multiple comparison. Bars, SE; *, P < 0.01 vs. Group A and < 0.05 vs. Group C; †, P < 0.01 vs. Group C.

Article Snippet: Then, sections were incubated with a mouse monoclonal anti-CD177 antibody (clone 4C4, diluted 1:100, Abnova, Taipei, Taiwan).

Techniques: Microarray, Infection, Quantitative RT-PCR, Expressing, Control, Comparison

Journal: BMC Gastroenterology

Article Title: Gene expression analysis of a Helicobacter pylori -infected and high-salt diet-treated mouse gastric tumor model: identification of CD177 as a novel prognostic factor in patients with gastric cancer

doi: 10.1186/1471-230X-13-122

Figure Lengend Snippet: CD177 expression in gastric carcinomas and its correlation with clinicopathological factors

Article Snippet: Then, sections were incubated with a mouse monoclonal anti-CD177 antibody (clone 4C4, diluted 1:100, Abnova, Taipei, Taiwan).

Techniques: Expressing

Journal: BMC Gastroenterology

Article Title: Gene expression analysis of a Helicobacter pylori -infected and high-salt diet-treated mouse gastric tumor model: identification of CD177 as a novel prognostic factor in patients with gastric cancer

doi: 10.1186/1471-230X-13-122

Figure Lengend Snippet: Multivariate analysis of prognostic factos in patients with gastric cancer using Cox proportional hazard model

Article Snippet: Then, sections were incubated with a mouse monoclonal anti-CD177 antibody (clone 4C4, diluted 1:100, Abnova, Taipei, Taiwan).

Techniques: Expressing

Journal: BMC Gastroenterology

Article Title: Gene expression analysis of a Helicobacter pylori -infected and high-salt diet-treated mouse gastric tumor model: identification of CD177 as a novel prognostic factor in patients with gastric cancer

doi: 10.1186/1471-230X-13-122

Figure Lengend Snippet: Regulated genes by combination of H. pylori infection and high-salt diet in mouse gastric mucosa

Article Snippet: Then, sections were incubated with a mouse monoclonal anti-CD177 antibody (clone 4C4, diluted 1:100, Abnova, Taipei, Taiwan).

Techniques: Infection, Transformation Assay, Membrane, Retroviral, Sequencing

Journal: PLoS ONE

Article Title: Sinomenine Protects against Lipopolysaccharide-Induced Acute Lung Injury in Mice via Adenosine A 2A Receptor Signaling

doi: 10.1371/journal.pone.0059257

Figure Lengend Snippet: Neutrophil infiltration into lung tissue was evaluated at 24 hour after acute lung injury by immunofluorescence using a CD177 primary antibody and a FITC-conjugated secondary antibody. Mice treated intratracheally with 40 µl PBS served as controls. (A) CD177-positive cells in the lung of control injured mice. (B) Cell counting and statistical analysis. * p <0.01 compared to the injured group without SIN treatment (0 mg/kg SIN treatment); # p <0.01 compared to the two indicated groups; NS: no significant difference between the two groups. (n = 8∼10 mice per group).

Article Snippet: Briefly, the frozen sections of injured lung tissue were fixed in acetone for 10 min, followed by incubation with rabbit anti-mouse CD177 antibody (Santa Cruz, CA, USA, 1:500), which labels neutrophils, on individual slides for 30 min at 37°C.

Techniques: Immunofluorescence, Control, Cell Counting

Journal: PLoS ONE

Article Title: Sinomenine Protects against Lipopolysaccharide-Induced Acute Lung Injury in Mice via Adenosine A 2A Receptor Signaling

doi: 10.1371/journal.pone.0059257

Figure Lengend Snippet: Thirty minutes before LPS injection to induce acute lung injury, 120 mg/kg SIN was administered to A 2A R KO mice. At 24 hour after ALI, neutrophil infiltration was detected by immunofluorescence using a CD177 primary antibody and a FITC-conjugated secondary antibody, and the protein expression levels of the inflammatory cytokines TNF-α and IL-1β were assayed by ELISA. The normal A 2A R KO mice intratracheally treated with 40 µl PBS were served as the control. (A) TNF-α protein levels. (B) IL-1β protein levels. (C) CD177-positive cells in the murine lung tissue. (D) Cell counting and statistical analysis. NS: no significant difference between the two groups. (n = 8∼10 mice per group).

Article Snippet: Briefly, the frozen sections of injured lung tissue were fixed in acetone for 10 min, followed by incubation with rabbit anti-mouse CD177 antibody (Santa Cruz, CA, USA, 1:500), which labels neutrophils, on individual slides for 30 min at 37°C.

Techniques: Injection, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Control, Cell Counting

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: Amino acid sequences of peptides that bind human and mouse CD177.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques:

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: Quantitative phage peptide binding assay.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: CHO cells expressing human CD177 were incubated for 3 h at 37°C with 75 μg/ml Peptide H-HPLNs. Cells were fixed and permeabilized, and CD177 was detected using a mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The HPLN particles are fluorescent red. As a negative control, HPLN particles displaying a scrambled peptide were used. The experiment was carried out twice, with at least 100 cells examined in each experiment. A direct correlation between the expression level of CD177 (based on fluorescence intensity) and the Peptide H-HPLN signal could be observed. The cell in the lower panel shows a typical cell distribution of CD177 and Peptide H-HPLNs after 3 h incubation. In contrast, the two cells shown in the upper panel are characteristic of the CD177 staining in the absence of Peptide H-HPLNs and in the presence of Scrambled Peptide H-HPLNs. Scale bar, 50 μm.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Expressing, Incubation, Negative Control, Fluorescence, Staining

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: CHO cells expressing human CD177 were incubated for 3 h and 17 h at 37°C with 75 μg/ml Peptide H-HPLN particles. Cells were fixed and permeabilized, and the lysosomes were stained with a mouse anti-hamster LAMP2 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. The selected micrographs are representative of more than 100 cells with similar labeling patterns. Scale bar, 50 μm.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Expressing, Incubation, Staining, Labeling

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: CHO cells expressing mouse CD177 containing an HA-tag were incubated with 75 μg/ml Peptide M-HPLN particles for 2 h on ice to allow binding. Cells were then washed and kept on ice or warmed to 37°C for 1 h. Cells were then treated with subtilisin to remove surface-bound Peptide M-HPLN particles, or treated with buffer alone. After fixation and permeabilization, mouse CD177-HA was stained with a mouse anti-HA antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. Most cells out of more than 100 cells visualized on the slides had a similar staining pattern as those seen in these micrographs. Scale bar, 50 μm.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Expressing, Incubation, Binding Assay, Staining

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: Peptide H-HPLN particles were incubated with whole human blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. Scrambled Peptide H-HPLN particles were used as a negative control. The experiment was carried out four times using blood from four different donors. In each case, slides with several hundred cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Incubation, Staining, Negative Control

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: Peptide M-HPLN particles were incubated with whole mouse blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with rabbit anti-mouse CD177 polyclonal antibody and Alexa Fluor 488 goat anti-rabbit secondary antibody. Peptide H-HPLN particles were used as a negative control. The experiment was carried out twice. The micrographs represent the results from >100 cells. Scale bar, 10 μm.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Incubation, Staining, Negative Control

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: Leukocytes were incubated on ice in the presence or absence of anti-CD177 antibody, washed and incubated on ice in the presence or absence of Peptide H-HPLN and Alexa Fluor 488 goat anti-mouse antibody. A. Forward and side scatter plot of human leukocytes (and remaining red blood cells) with the neutrophil population shown in a circle. B. FL-1 histogram of CD177 expression. Left panel: Total cell population in the absence and presence of anti-CD177 antibody (1° Ab) and Alexa Fluor 488 goat anti-mouse antibody (2° Ab). Right panel: Gated neutrophil population in the absence or presence of anti-CD177 antibody (1° Ab) and Alexa Fluor 488 goat anti-mouse antibody (2° Ab). C. FL2 plot showing Peptide H-HPLN binding. Total cell population in the absence or presence of Peptide H-HPLNs (left panel) and gated neutrophil population in the absence and presence of Peptide H-HPLNs (right panel). D. FL1-A and FL2-A plot showing fluorescence of the total cell population (left panel) and gated neutrophil population (right panel). The experiment was carried out twice with similar results. The blood sample shown had a higher percentage of CD177-positive neutrophils than the blood sample from the other donor.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Incubation, Expressing, Binding Assay, Fluorescence

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: Purified human neutrophils were incubated with Peptide H-HPLNs at 37°C with aliquots taken at 0, 15, 30, 60 and 120 min. Cells were rinsed with PBS, centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once using blood from a donor with a high CD177 expression level. Hundreds of cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.

Article Snippet: Rabbit polyclonal anti-mouse CD177 antibody, clone 1171A, catalog # MAB8186, was from R&D Systems.

Techniques: Purification, Incubation, Staining, Expressing