Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet: CD163 + macrophages accumulate in the lung in severe COVID-19 (A) Overview of study design and analyses. CT, computed tomography; BAL, bronchoalveolar lavage; scRNA-seq, single-cell RNA sequencing; snRNA-seq, single-nucleus RNA sequencing; IHC, immunohistochemistry; IF, immunofluorescence microscopy; MELC, multi-epitope ligand cartography; EM, electron microscopy; VCin, inspiratory vital capacity; PBMC, peripheral blood mononuclear cells; IAV, Influenza A virus. (B) Postmortem analysis of consecutive histological sections of non-COVID-19 (left) and COVID-19 autopsy lung samples (right) by hematoxylin and eosin (H&E; top) and CD68 IHC (bottom). Scale bar, 100 μm. (C) IF of CD68 (green) and CD163 (red) in lung tissue autopsy samples of COVID-19 patients and non-COVID-19 controls. Arrows indicate CD68 + CD163 – macrophages, and arrowheads indicate CD68 + CD163 + macrophages. Scale bar, 20 μm. (D) Quantification of CD68 + macrophage density (left) and the proportion of CD163 + macrophages (right) in lung autopsy samples from fifteen donors (as in C). Mann-Whitney test; ∗ p < 0.05. (E) Representative images of consecutive histological sections of lung autopsy samples. H&E (left), CD68 IHC (middle), and SARS-CoV-2 RNA-FISH (right). Arrowheads indicate SARS-CoV-2 RNA-positive macrophages. Scale bars, 50 μm, 25 μm. RNA-FISH, RNA-fluorescence in situ hybridization. (F) Lung autopsy samples of 9 COVID-19 patients were analyzed by MELC with a panel of 22 markers on 19 fields of view (FOVs). Two-dimensional embedding computed by UMAP on 9,684 computationally identified CD45 positive cells (T cells, CD3 + ; B cells, CD20 + ; NK cells, CD56 + ; neutrophils, MRP14 + /CD66b + ; monocytes, MRP14 + /CCR2 + ; macrophages, MRP14 + /HLA-DR + ). (G) Relative proportion (of total CD45 + cells) of cell types in all 19 FOVs (left), and average cell numbers (summary, right).

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Computed Tomography, RNA Sequencing Assay, Immunohistochemistry, Immunofluorescence, Microscopy, Electron Microscopy, MANN-WHITNEY, Fluorescence, In Situ Hybridization

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet:

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Immunohistochemistry, Plasmid Preparation, Recombinant, Staining, Lysis, Protease Inhibitor, Mass Spectrometry, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 162 Dy , CD66b , 80H3 , Fluidigm 3162023B.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Effects of acute hypoxia on the development of giant phagocytes (G ϕ ). Freshly isolated PMN were exposed for 24 h to intermittent hypoxia (IH, 56 cycles), sustained hypoxia (SH), or normoxia (N) and then cultured at normoxia for additional six days. Cytospins were prepared and analyzed by confocal microscopy (see Materials and Methods). Nuclei were stained with DAPI (blue). (a) Fixed cytospins were stained with anti-CD66b Abs and (b) neutrophil elastase (NE). (c) Isotype controls: fixed cytospins were stained with mouse IgG2 followed by 1/400 CF 647 goat anti-mouse IgG (red) staining. (d) Viable cells were stained with LysoTracker before fixation (see Materials and Methods). Arrow indicates large phagolysosomes. Representative data out of 3 independent experiments.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques: Isolation, Cell Culture, Confocal Microscopy, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

doi: 10.1155/2016/9636937

Figure Lengend Snippet: Expression of NADPH oxidase subunits in giant phagocytes (G ϕ ) and the effects of NAC on their expression. Freshly isolated PMN were exposed for 24 h to intermittent hypoxia (IH, 56 cycles), sustained hypoxia (SH), or normoxia (N) and then cultured at normoxia for additional six days. NAC (20 μ M) was added to PMN cultures 10 min prior to exposing to N, IH, or SH. Equal volumes of DMSO were added as a negative control. Cytospins were prepared and analyzed by confocal microscopy (see Materials and Methods). The developed G ϕ were stained by double immunofluorescence: (a) for CD66b (red) and gp91- phox (green) in untreated and NAC-treated G ϕ and (b) for gp91- phox (green) and p22- phox (red) in untreated and NAC-treated G ϕ . Nuclei were stained with DAPI. Representative photomicrographs out of 3 independent experiments.

Article Snippet: For intracellular staining, cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Israel) in PBS, at room temperature for 10 min. After blocking with 10% normal goat serum in RPMI-1640, cells were incubated overnight at 4°C using the following primary Abs (dilution 1 : 100) or the corresponding isotype controls: mouse monoclonal anti-CD66b Abs (80H3, AbD Serotec, Oxford, UK) and anti-cytochrome b-245 light chain (p22- phox identification, Clone 44.1, BioLegend, San Diego, CA), rabbit polyclonal anti-neutrophil elastase (NE) (Calbiochem, San Diego, CA), anti-LC3B Abs (Sigma, Israel), and anti-Nox2/gp91- phox Abs (ab131083, Abcam, UK).

Techniques: Expressing, Isolation, Cell Culture, Negative Control, Confocal Microscopy, Staining, Immunofluorescence

Journal: Cell

Article Title: Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19

doi: 10.1016/j.cell.2020.08.002

Figure Lengend Snippet:

Article Snippet: CD66b , Fluidigm , Pro# 3152011B.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Staining, Functional Assay, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Peritumoural neutrophils negatively regulate adaptive immunity via the PD-L1/PD-1 signalling pathway in hepatocellular carcinoma

doi: 10.1186/s13046-015-0256-0

Figure Lengend Snippet: Neutrophils predominantly infiltrated in the peritumoural tissue in patients with HCC. a , b Representative images were shown for CD66b and CD3 immunohistochemical staining of tumour tissues from patients with HCC. A high density of CD66b + neutrophils is correlated with a low density of CD3 + T cells in the tumour tissue. c Infiltrating CD66b + neutrophils and CD3 + T cells in intratumoural tissue and peritumoural tissues that were analysed by paired t -Test ( P < 0.001 for both). d The neutrophil/T cell ratio (NLR) in intratumoural tissue and peritumoural tissues that were analysed by paired t -Test ( P < 0.001 for both). e Correlation of the number of CD66b + neutrophils and CD3 + T cells in peritumoural tissues and intratumoural tissues in patients with HCC as analyzed by Pearson correlation analysis and linear regression analysis ( r = −0.7902, P < 0.001 and r = 0.2890, P = 0.0974, respectively)

Article Snippet: The following antibodies were used as primary antibodies: anti-human CD66b (Anaspec, CA, USA), anti-human CD3 (Gene Tech, Shanghai, China), anti-human PD-L1 and anti-human PD-1 (BD Bioscience, San Jose, USA).

Techniques: Immunohistochemical staining, Staining