Journal: Cell Reports Medicine

Article Title: Targeting mitochondrial complex I of CD177 + neutrophils alleviates lung ischemia-reperfusion injury

doi: 10.1016/j.xcrm.2025.102140

Figure Lengend Snippet: Single-cell transcriptional profiling of lung ischemia-reperfusion injury (A) Schematic diagram illustrating the experimental design. Left lung tissues and blood samples of mice subjected to lung ischemia (1 h) followed by reperfusion (3 h) were collected. Subsequent single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses were performed. Clinical validation involved examining CD177 + neutrophils collected pre- and post-transplantation in human lung recipients. Functional proteomics identified increased mitochondrial complex I activity in CD177 + neutrophils. Treatment with IACS-010759 (1 mg/kg), a complex I inhibitor, alleviated lung IRI. (B) Uniform manifold approximation and projection (UMAP) plot displaying 43,852 immune cells from lung tissues under sham ( n = 4 mice) and IRI ( n = 6 mice) conditions. These immune cells were categorized into 5 major cell types. (C) The differentially expressed genes (DEGs) of the 5 major cell clusters are listed. Myeloid cells show the highest number of DEGs. (D) Bubble heatmap highlighting genes significantly increased across 10 major subclusters of immune cells in lung IRI. Average fold change (FC) and significance (−Log 10 -adjusted p value) are indicated by dot size and color, respectively. (E) Relative gene expression scores of post- vs. pre-transplantation samples in human lung transplant recipients. (F) UMAP plot displaying 2,973 lung-associated neutrophils (LANs) subdivided into 5 cell types (LAN1–LAN5). (G) Heatmap of the top 6 differential genes for LAN1–LAN5. (H) Feature plots of selected marker genes ( Cebpb , Ccrl2 , Cd177 , Camp , and Itgal ) showing high expression of Cd177 in specific neutrophil sub-clusters. (I) Cell proportions of LAN1–LAN5 in IRI versus sham control groups showing LAN3 was significantly increased after lung IRI. (J) Gene Ontology enrichment analysis of LAN1–LAN3 neutrophil clusters, indicating that LAN3 is related to neutrophil activation. NK, natural killer; Neut, neutrophil; AMϕ, alveolar macrophage; Mo, monocyte. Statistical significance was assessed by two-sided Wilcoxon test adjusted using Bonferroni method in (C), (D), and (G), two-sided Wilcoxon test in (H), and Fisher’s exact test in (J). ns, not significant; ∗∗∗ p < 0.001.

Article Snippet: The Cd177 flox/flox ; Ly6g Cre mice (Male, 8–10 weeks) were obtained from Cyagen Company (Jiangsu, China).

Techniques: RNA Sequencing, Biomarker Discovery, Transplantation Assay, Functional Assay, Activity Assay, Gene Expression, Marker, Expressing, Control, Activation Assay

Journal: Cell Reports Medicine

Article Title: Targeting mitochondrial complex I of CD177 + neutrophils alleviates lung ischemia-reperfusion injury

doi: 10.1016/j.xcrm.2025.102140

Figure Lengend Snippet: Enriched Cd177 + neutrophils are key contributors to lung ischemia-reperfusion injury in the mouse model (A) Nucleic acid staining and watershed-based segmentation for single-cell analysis. A representative image shows nuclei segmented using the watershed algorithm for spatial distribution analysis ( n = 2). (B) Spatial expression of the pan-immune marker Cd45 in lung tissue sections reveals regions of local immune cell infiltration. (C) Spatial mapping of Cd177 expression in lung tissues after IRI. Higher-magnification images (right) show colocalization of Cd177 with the inflammatory genes Pglyrp1 and Ltf . (D) Uniform manifold approximation and projection (UMAP) visualization showing Ly6g + Cd177 + neutrophil populations (red) compared with Ly6g + Cd177 − neutrophils (blue). The bubble heatmap on the right demonstrates enrichment of inflammatory Gene Ontology terms in Ly6g + Cd177 + cells. (E) Representative immunofluorescence images from the mouse left lung for Ly6G (green), CD177 (red), and citrullinated histone H3 (Cit-H3, white) showing NET formation in CD177 + neutrophils. Scale bar: 20 μm. (F) Reactive oxygen species (ROS) production was higher in CD177 + compared to CD177 − neutrophils from human samples (left) and in Cd177 flox/flox neutrophils compared to Cd177 flox/flox ; Ly6g Cre neutrophils from mice (right), with or without PMA stimulation. ( n = 5 per group). Neut, neutrophils; UT, untreated. (G) Quantification of MPO-DNA complexes showing increased NET formation in CD177 + compared to CD177 − human neutrophils (left) and in Cd177 flox/flox compared to Cd17 7 flox/flox ; Ly6g Cre mouse neutrophils (right) under PMA stimulation. Neut, neutrophils; UT, untreated. ( n = 5 per group). (H) Representative H&E staining of lung sections from Cd177 flox/flo x and Cd177 flox/flox ; Ly6g Cre mice under sham and IRI conditions. Quantification of acute lung injury scores (right) demonstrates significant injury in Cd177 flox/flox mice and minimal injury in Cd177 flox/flox ; Ly6g Cre mice after lung IRI. Scale bar: 50 μm. ( n = 6 per group). (I) Immunofluorescence staining revealing reduced NET infiltration (Ly6G, Cit-H3, and DNA/H1) in lung tissues of Cd177 flox/flox ; Ly6g Cre mice post-lung IRI. Scale bar: 20 μm. Data are shown as mean ± SD. Statistical significance was assessed by a two-sided Wilcoxon test adjusted with the Bonferroni method in (F), (G), and (H) and Fisher’s exact test in (D). ns, not significant; ∗∗∗ p < 0.001.

Article Snippet: The Cd177 flox/flox ; Ly6g Cre mice (Male, 8–10 weeks) were obtained from Cyagen Company (Jiangsu, China).

Techniques: Staining, Single-cell Analysis, Expressing, Marker, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Targeting mitochondrial complex I of CD177 + neutrophils alleviates lung ischemia-reperfusion injury

doi: 10.1016/j.xcrm.2025.102140

Figure Lengend Snippet: CD177 + neutrophils predict grade 3 primary graft dysfunction (A) UMAP plot displaying four distinct mouse peripheral blood neutrophil subsets (PBN1–PBN4) under sham and ischemia-reperfusion injury (IRI) conditions ( n = 3 per group). (B) Boxplot quantifying PBN3 proportions in IRI vs. sham groups, showing a significant increase in PBN3 cells in the IRI group. (C) Bubble heatmap showing the expression of marker genes in mouse PBN1–4 and mouse lung-associated neutrophils 1–3 (LAN1–3). Both PBN3 and LAN3 exhibit high Cd177 , Ngp , Camp , and Ltf expressions, indicating an activated phenotype contributing to IRI pathology. (D) Violin plots illustrating the bone marrow proximity scores of mice PBN1–4 and LAN1–3. PBN3 and LAN3 have notably high scores, indicating recent mobilization from the bone marrow. (E and F) Pseudotime analyses of murine peripheral blood neutrophil subsets (PBN1–4). (E) CytoTrace and (F) Monocle 3 highlight a neutrophil differentiation trajectory, with PBN3 cells occupying an earlier differentiation state. (G and H) Boxplots showing (G) the neutrophil-to-lymphocyte ratio across PGD 0–2 and PGD 3 groups ( n = 79 vs. 25), demonstrating no significant difference, and (H) a significant increase in CD177 + neutrophil proportions in PGD 3 vs. PGD 0–2. (I) Receiver operating characteristic (ROC) curve displaying the diagnostic performance of the change in the proportion of CD177 + neutrophils (between post-4 h and pre-transplantation) in predicting grade 3 PGD within 72 h. The ROC analysis demonstrates high diagnostic accuracy (AUC = 0.871; 95% CI: 0.764–0.951). Statistical significance was assessed by a two-sided Wilcoxon test adjusted with the Bonferroni method in (B), (D), (G), and (H). ns, not significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The Cd177 flox/flox ; Ly6g Cre mice (Male, 8–10 weeks) were obtained from Cyagen Company (Jiangsu, China).

Techniques: Expressing, Marker, Diagnostic Assay, Transplantation Assay

Journal: Cell Reports Medicine

Article Title: Targeting mitochondrial complex I of CD177 + neutrophils alleviates lung ischemia-reperfusion injury

doi: 10.1016/j.xcrm.2025.102140

Figure Lengend Snippet: CD177 + neutrophils exhibit enhanced mitochondrial oxidative phosphorylation and electron transport activity (A) scMetabolism analysis for lung-associated neutrophils 1–3 (LAN1–3) showing oxidative phosphorylation (OXPHOS) pathway was significantly enriched in the LAN3 subset. (B) Quantification of electron transport score (left) and OXPHOS score (right) in LAN1–3 demonstrates that the LAN3 subset has the highest values for both. (C and D) Gene Ontology (GO) enrichment analysis of upregulated pathways in human CD177 + neutrophils (C, n = 4 per group) and Cd177 flox/flox mouse neutrophils (D, n = 3 per group), based on proteomic data. OXPHOS and respiratory complex I pathways were enriched. (E) Heatmap showing increased expression of mitochondrial proteins associated with OXPHOS, the electron transport chain, and the respiratory chain complexes in human CD177 + neutrophils compared to CD177 − neutrophils ( n = 4 per group). (F) Representative transmission electron microscopy (TEM) images depicting the mitochondrial ultrastructure of human CD177 + and CD177 − neutrophils. The quantitative analysis of mitochondrial cristae width (right) reveals a significant increase in cristae width in CD177 + neutrophils ( n = 8 per group). (G) Oxygen consumption rate (OCR) analysis of human CD177 + and CD177 − neutrophils under basal and pharmacologically modulated conditions demonstrates enhanced mitochondrial oxidative phosphorylation in CD177 + cells. Treatment with the mTOR agonist MHY1485 markedly enhances OXPHOS activity in CD177 − neutrophils, while inhibition of mTOR signaling via AZD8055 suppresses OXPHOS in CD177 + cells, indicating mTOR-dependent regulation of mitochondrial respiration. Data represent n = 9 replicates per group. (H) Blue native PAGE and immunoblot assay of mitochondrial complex subunits (CI–V) in human CD177 + and CD177 − neutrophils. Quantification (right) shows elevated expression of NDUFS1 (a marker for mitochondrial complex I) in CD177 + neutrophils. Data are shown as mean ± SD ( n = 4 per group). (I) Immunofluorescence staining shows a marked upregulation of MitoSOX (red) in human CD177 + neutrophils compared to CD177 − neutrophils. Scale bar: 10 μm. (J) Immunofluorescence staining of human neutrophils reveals robust colocalization of ILK (red), CD177 (green), and CD11b (white) in CD177 + neutrophils. Scale bar: 10 μm. (K) Immunoblot assay of ILK protein expression in human CD177 + and CD177 − neutrophils after CD11b immunoprecipitation (IP). β-actin serves as a loading control for the input. (L) Immunoblot assay of AKT, phosphorylated AKT (p-AKT), mTOR, phosphorylated mTOR (p-mTOR), and NDUFS1 expression in CD177 + and CD177 − neutrophils with or without AZD8055 and MHY1485 treatment. CD177 + cells show higher p-AKT and p-mTOR levels vs. CD177 − cells. AZD8055 (mTOR inhibitor) reduces p-mTOR and NDUFS1 in CD177 + cells, while MHY1485 (mTOR agonist) increases these proteins in CD177 − cells. Data are shown as mean ± SD. Statistical significance assessed by a two-sided Wilcoxon test adjusted with the Bonferroni method (B and F). ns, not significant; ∗∗∗ p < 0.001.

Article Snippet: The Cd177 flox/flox ; Ly6g Cre mice (Male, 8–10 weeks) were obtained from Cyagen Company (Jiangsu, China).

Techniques: Phospho-proteomics, Activity Assay, Expressing, Transmission Assay, Electron Microscopy, Inhibition, Blue Native PAGE, Western Blot, Marker, Immunofluorescence, Staining, Immunoprecipitation, Control

Journal: Cell Reports Medicine

Article Title: Targeting mitochondrial complex I of CD177 + neutrophils alleviates lung ischemia-reperfusion injury

doi: 10.1016/j.xcrm.2025.102140

Figure Lengend Snippet: Mitochondrial complex I inhibitor IACS-010759 alleviates lung ischemia-reperfusion injury (A) OCR analysis showing mitochondrial respiration in CD177 + and CD177 − neutrophils and CD177 + neutrophils treated with 25 or 50 nM IACS-010759. Quantification of ATP production and basal respiration indicates a significant reduction of oxidative phosphorylation (OXPHOS) after IACS-010759 treatment ( n = 9 per group). (B) Representative images of left lung H&E staining (left) and lung injury scores (right) showing IACS-010759 (1 mg/kg) treatment reduced lung injury in the left hilar ligation/reperfusion mouse model. Scale bar: 50 μm ( n = 5 per group). (C) UMAP visualization of scRNA-seq data from sorted neutrophils in lungs of IACS-treated ( n = 4) and untreated mice ( n = 3) under left hilar ligation/reperfusion. (D) Quantification of Cd177 + neutrophils in the IACS-treated versus untreated groups following lung IRI. IACS treatment significantly reduces the proportion of Cd177 + neutrophils. (E) Violin plots illustrating the OXPHOS (left) and electron transport chain (right) scores of Cd177 + neutrophils in IRI lungs, showing a significant decrease after IACS treatment. (F) Schematic of the orthotopic left lung transplantation rat model with prolonged cold ischemia. (G) IACS treatment reduced lung injury, as evidenced by H&E staining (left) and lung injury scores (right) from sham rats and allografts treated with vehicle or IACS-010759 (1 mg/kg) following prolonged ischemia reperfusion ( n = 6 per group). Scale bar: 50 μm. (H) Immunofluorescence staining for neutrophil extracellular traps (Ly6G, DNA/H1, and Cit-H3) in lung tissues of sham and IRI rats with or without IACS treatment. Scale bar: 20 μm. (I) Lung injury indicators and pulmonary function (airway compliance, airway resistance, PaO 2 , and PaCO 2 ) measured in each group showing that IACS treatment reduced lung injury in the orthotopic left lung transplant rat model ( n = 5 per group). Data are shown as mean ± SD. Statistical significance was assessed by a two-sided Wilcoxon test adjusted with the Bonferroni method (A, B, D, E, G, and I). ns, not significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The Cd177 flox/flox ; Ly6g Cre mice (Male, 8–10 weeks) were obtained from Cyagen Company (Jiangsu, China).

Techniques: Phospho-proteomics, Staining, Ligation, Transplantation Assay, Immunofluorescence

Journal: Experimental and therapeutic medicine

Article Title: Proteinase 3 expression on the neutrophils of patients with paroxysmal nocturnal hemoglobinuria.

doi: 10.3892/etm.2017.5662

Figure Lengend Snippet: Figure 1. PR3 and NB1 expression on the neutrophil plasma membranes of patients with PNH or PNH‑AA is decreased. The expression of PR3 and NB1 were detected by flow cytometry in (A) PNH/PNH‑AA patients and (B) healthy controls (presented as the mean ± standard error of the mean). (A1 and B1) The neutrophils were gated as R1 and then (A2 and B2) NB1 expression was investigated on CD59‑/CD59+ neutrophils. (A3 and B3) PR3 and NB1 were demon strated to be expressed on CD59‑/CD59+ neutrophils. (C) Quantification and analysis of the flow cytometry results. (C1 and C2) The results demonstrated that the expression of NB1 and PR3 on CD59‑ neutrophils significantly decreased compared with CD59+ neutrophils in patients with PNH/PNH‑AA and the healthy controls. (C3) No correlation was identified between PR3 and NB1 expression in patients with PNH/PNH‑AA. Furthermore, the expression of these two proteins were measured by immunofluorescence. PR3 was partially expressed on CD59‑ neutrophils of (D1) patients with PNH/PNH‑AA compared with (D2) healthy controls, while no NB1 expression was identified on CD59‑ neutrophils of (E1) patients with PNH/PNH‑AA compared with (E2) healthy controls. SSC, side‑scattered light; FSC, forward‑scattered light; CD, cluster of differentiation; NB1, CD177 antigen; APC, allophycocyanin; PE, phycoerythrin; PR3, proteinase 3; FITC, fluorescein isothiocyanate; PNH, paroxysmal nocturnal hemoglobinuria; AA, aplastic anemia.

Article Snippet: Briefly, 100 μl of diluted (1:100) capture antibodies directed against PR3 (Human proteinase-antineutrophil cytoplasmic antibody; PR3-ANCA ELISA kit; cat no. fk1344Y; R&D Systems, Inc, Minneapolis, MN, USA), NB1 (CD177 ELISA kit; cat no. EH1752; Cusabio Biotech Co., Ltd., Wuhan, China) or PAR1 (Human Protease Activated Receptor 1 ELISA kit; cat no. SEC939Hu; Cloud-Clone Corp., Katy, TX, USA) were added to each well and the plates were incubated at 4 ̊C overnight.

Techniques: Expressing, Clinical Proteomics, Flow Cytometry, Immunofluorescence

Journal: Experimental and therapeutic medicine

Article Title: Proteinase 3 expression on the neutrophils of patients with paroxysmal nocturnal hemoglobinuria.

doi: 10.3892/etm.2017.5662

Figure Lengend Snippet: Figure 3. PNH clones exhibit no significant difference in mRNA and protein levels of PR3 and NB1 compared with neutrophils from the controls. (A) The neutrophils were isolated from the blood of patients with PNH/PNH‑AA; the purity, detected by flow cytometry, was >85%. (B) CD59‑ neutrophils were sorted by magnetic‑activated cell sorting and the purity was determined to be >90%. No significant differences in the mRNA expression of (C) PR3 or (D) NB1 were identified between patients with PNH/PNH‑AA and the healthy controls (presented as the mean ± standard error of the mean). (E) No notable differences in PR3 protein expression were identified between patients with PNH/PNH‑AA and the healthy controls. SSC, side‑scattered light; FSC, forward‑scattered light; CD, cluster of differentiation; NB1, CD177 antigen; PE, phycoerythrin; PR3, proteinase 3; PNH, paroxysmal nocturnal hemoglobinuria; AA, aplastic anemia.

Article Snippet: Briefly, 100 μl of diluted (1:100) capture antibodies directed against PR3 (Human proteinase-antineutrophil cytoplasmic antibody; PR3-ANCA ELISA kit; cat no. fk1344Y; R&D Systems, Inc, Minneapolis, MN, USA), NB1 (CD177 ELISA kit; cat no. EH1752; Cusabio Biotech Co., Ltd., Wuhan, China) or PAR1 (Human Protease Activated Receptor 1 ELISA kit; cat no. SEC939Hu; Cloud-Clone Corp., Katy, TX, USA) were added to each well and the plates were incubated at 4 ̊C overnight.

Techniques: Clone Assay, Isolation, Flow Cytometry, FACS, Expressing

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

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: Mouse ( Mus musculus) CD177 cDNA was obtained from OriGene Technologies in the pCMV6-Kan/Neo plasmid (catalog number MC201519; GenBank Accession BC027283).

Techniques: Purification, Incubation, Staining, Expressing

Journal: Diagnostics

Article Title: Comparison of Two Spectral-Domain Optical Coherence Tomographs for Macular Thickness in Romanian Children Reveals Device-Dependent Normative Data and Significant Gender Differences

doi: 10.3390/diagnostics16040609

Figure Lengend Snippet: ( a ) Spectralis SD-OCT scan report of macular thickness. ( b ) SOCT Copernicus REVO macular thickness scan report [personal database].

Article Snippet: Similarly, Jammal et al. [ ], examining 144 Jordanian children (ages 6–16 years) using the Primus SD-OCT, found that boys had significantly higher CST than girls (adjusted mean 250.68 μm [95% CI: 246.68, 254.68] vs. 243.06 μm [95% CI: 239.29, 246.84]; p = 0.008).

Techniques:

Journal: Diagnostics

Article Title: Comparison of Two Spectral-Domain Optical Coherence Tomographs for Macular Thickness in Romanian Children Reveals Device-Dependent Normative Data and Significant Gender Differences

doi: 10.3390/diagnostics16040609

Figure Lengend Snippet: SD-OCT measurements based on gender.

Article Snippet: Similarly, Jammal et al. [ ], examining 144 Jordanian children (ages 6–16 years) using the Primus SD-OCT, found that boys had significantly higher CST than girls (adjusted mean 250.68 μm [95% CI: 246.68, 254.68] vs. 243.06 μm [95% CI: 239.29, 246.84]; p = 0.008).

Techniques:

Journal: Diagnostics

Article Title: Comparison of Two Spectral-Domain Optical Coherence Tomographs for Macular Thickness in Romanian Children Reveals Device-Dependent Normative Data and Significant Gender Differences

doi: 10.3390/diagnostics16040609

Figure Lengend Snippet: Age influence on SD-OCT measurements.

Article Snippet: Similarly, Jammal et al. [ ], examining 144 Jordanian children (ages 6–16 years) using the Primus SD-OCT, found that boys had significantly higher CST than girls (adjusted mean 250.68 μm [95% CI: 246.68, 254.68] vs. 243.06 μm [95% CI: 239.29, 246.84]; p = 0.008).

Techniques:

Journal: Diagnostics

Article Title: Comparison of Two Spectral-Domain Optical Coherence Tomographs for Macular Thickness in Romanian Children Reveals Device-Dependent Normative Data and Significant Gender Differences

doi: 10.3390/diagnostics16040609

Figure Lengend Snippet: SD-OCT measurements between the two measuring devices. * p = 0.02, ** p < 0.01.

Article Snippet: Similarly, Jammal et al. [ ], examining 144 Jordanian children (ages 6–16 years) using the Primus SD-OCT, found that boys had significantly higher CST than girls (adjusted mean 250.68 μm [95% CI: 246.68, 254.68] vs. 243.06 μm [95% CI: 239.29, 246.84]; p = 0.008).

Techniques:

Journal: Frontiers in Immunology

Article Title: Targeted nanoparticles modify neutrophil function in vivo

doi: 10.3389/fimmu.2022.1003871

Figure Lengend Snippet: Murine Neutrophil-Specific LQI-Peptide binding via CD177. (A) Schematic representation of the biotin-LQI-tetramer. (B) FACS analysis comparing the binding of LQI tetramer (left) and 16-LQI (right) to WBC following 20 min. incubation with 1μM of each. (C) Quantification of LQI tetramer (blue) or 16-LQI (red) binding to neutrophils and other WBC. (D) Mouse neutrophils were incubated with the LQI-tetramer, the 16-LQI complex and the 16-Cont. (human neutrophil binding peptide - KFP) complex in increasing concentration. The samples were then analyzed by flow cytometry and the presented shows the fraction of Cy3 + neutrophils (Ly6G + ). The 16-LQi and the 16-Cont. were preincubated with SA-Cy3, the LQI-tetramer was first incubated with the cells and then incubated with SA-Cy3. (E) Quantification of FACS analysis of the extent of binding of 16-LQI (1μM) to circulating Ly6G + neutrophils from healthy Balb/C and C57BL/6 mice (n=3). (F) Silver staining of the gel plotting the proteins pulled down using naked (Cont.) and LQI-tetramer decorated (LQI-peptide) SA agarose beads. (G) Top ranking proteins enriched by LQI-tetramer-SA pull down. (H, I) Representative dot plots of isolated WBC stained with Ly6G and CD177 (H) and CD177 and 1μM LQI-tetramer (I) . (J) FACS analysis of 16-LQI binding to control HEK293T cells (Red), SA-Cy3 binding to HEK293T cells overexpressing the murine CD177 protein (Green) and CD177-overexpressing HEK293T cells were incubated with 16-LQI (blue). (K) Representative STORM imaging of a single neutrophil with staining of CD177 (green) and 16-LQI (red), overlay with brightfield (left) or DAPI staining of nucleus (right). (L) Quantification of CD177 and 16-LQI interaction for image depicted in (K) using the ImageJ Interaction Factor plugin . Error bars represent ± SEM. * p<0.05.

Article Snippet: Antibodies used in this study were the following: rat anti mouse Ly6G-violet 450 (Tonbo Biosciences), rat anti mouse Gr1-FITC (Tonbo Biosciences), rat anti mouse/human CD45-PE (BD Biosciences), rat anti mouse/human CD45-APC (Bio Legend), rat anti mouse/human CD11b-FITC (BD Biosciences), rat anti mouse CD177-Alexa Fluor 647 (R&D Systems), rat anti human CD177-APC (Bio Legend), mouse anti phage M13-PE (Santa Cruz), rabbit anti mouse/human pSmad2 (Cell Signaling), rabbit anti mouse/human pSmad3 (Abcam), rabbit anti mouse/human Smad2/3 (Cell Signaling), Annexin-V FITC (Biotium) and SA-Alexa Fluor 568 (Thermo Scientific).

Techniques: Binding Assay, Incubation, Concentration Assay, Flow Cytometry, Silver Staining, Isolation, Staining, Control, Imaging

Journal: Frontiers in Immunology

Article Title: Targeted nanoparticles modify neutrophil function in vivo

doi: 10.3389/fimmu.2022.1003871

Figure Lengend Snippet: Characterization of Human P-NP. (A) High ranking human neutrophil binding peptide sequences. (B) Binding of phages expressing different peptides to CD66b + neutrophils from different donors (colored circles). Open black circles represent an outlier excluded from the overall average. (C) Representative quantification of 16-KFP binding to neutrophils (SSC high , blue) and other WBC (SSC low , red) from a healthy donor. (D) 16-KFP binding and CD177 expression of neutrophils of healthy donors ( , , ), COPD patients ( , ) and lung cancer patients . Note the low neutrophil binding and low CD177 expression in healthy donor #2. (E) Correlation between CD177 expression and 16-KFP binding in different donors. (F) Binding of 16-KFP to neutrophils with increasing amounts of CD177 blocking antibody. (G) FACS analysis of human neutrophil binding of KFP-tetramer coated (right) and uncoated (middle) PLGA-Cy5 labelled NP in WBC from a healthy donor. (H) PMA induced ROS production in control neutrophils (Cont.), neutrophils treated with empty (Empty NP) or DPI-containing P-NP (DPI NP).

Article Snippet: Antibodies used in this study were the following: rat anti mouse Ly6G-violet 450 (Tonbo Biosciences), rat anti mouse Gr1-FITC (Tonbo Biosciences), rat anti mouse/human CD45-PE (BD Biosciences), rat anti mouse/human CD45-APC (Bio Legend), rat anti mouse/human CD11b-FITC (BD Biosciences), rat anti mouse CD177-Alexa Fluor 647 (R&D Systems), rat anti human CD177-APC (Bio Legend), mouse anti phage M13-PE (Santa Cruz), rabbit anti mouse/human pSmad2 (Cell Signaling), rabbit anti mouse/human pSmad3 (Abcam), rabbit anti mouse/human Smad2/3 (Cell Signaling), Annexin-V FITC (Biotium) and SA-Alexa Fluor 568 (Thermo Scientific).

Techniques: Binding Assay, Expressing, Blocking Assay, Control