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: bioRxiv

Article Title: Immune Checkpoint Molecules as Biomarkers of Staphylococcus aureus Bone Infection and Clinical Outcome

doi: 10.1101/2024.12.30.630837

Figure Lengend Snippet: Bone tissues surgically removed from PJI patient with S. aureus osteomyelitis were processed for histology and immunohistochemistry. (A) Representative 100x image (bar =100 μm) of a H&E-stained section is shown to illustrate the inflammatory cells within the region of interest (box). ( B-D) Parallel histology sections containing the region of interest were immunostained with labelled antibodies against CD3, PD1, S. aureus, TIM-3 (green), LAG-3, and CD66b, counter stained with DAPI, and representative fluorescent microscopy images are shown at 200x (bar = 100 μm). ( B ) Note CD3 + /PD1 + T cells detected in areas of S. aureus infection (white arrows). (C) Note CD3 + /TIM-3 + (white arrows) and CD3 + /LAG-3 + (yellow arrows) T cells at the site of S. aureus infection. (D) Note TIM-3 + /CD66b+ neutrophils at the site of infection (white arrows).

Article Snippet: Primary antibodies: The following antibodies were utilized for immunostaining: goat anti-CD3ε (clone M-20, sc-1127, RRID:AB_631128, Santa Cruz Biotechnology), mouse anti-PD-1 (10377-MM23, RRID:AB_2936309, Sino Biologicals), Rabbit anti-LAG3 (clone BLR027F, NBP2-76402, RRID:AB_3403543, Novus Biologicals), Mouse anti-TIM3/HAVCR2 (clone TIM3/4031, V8754-20UG, NSJ Bioreagents), Rabbit anti- S. aureus (PA1-7246, RRID:AB_561546, Thermo Fisher Scientific), and Mouse anti-CD66b (G10F5, NBP2-80664, RRID:AB_3096017, Novus Biologicals).

Techniques: Immunohistochemistry, Staining, Microscopy, Infection

Journal: Cancer Research

Article Title: Human Tumor–Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis

doi: 10.1158/0008-5472.CAN-23-2135

Figure Lengend Snippet: The ability of blood and tumor myeloid cells to perform ADT and kill EGFR hi A431 cells in the presence of anti-EGFR Abs under conditions representing solid human tumors. A, The frequencies of CD45 − EpCam + cells, CD14 + CD206 + HLA-DR hi TAM, and CD11b + CD66b + CD14 − TAN were analyzed by flow cytometry in digested lung tumors. Paired t test. B and C, Representative dot plots showing the levels of trogocytosis and killing activity mediated by blood monocytes ( B ) and PBNs ( C ) cocultured with PKH67 + A431 cells at the indicated E:T ratios in the presence of cetuximab and IgA anti-EGFR Abs, respectively, for 12 hours. Nonspecific human IgG1 or IgA2 isotype control Abs were used as a control. D and E, Cumulative flow cytometry results showing the ability of indicated FcR + effectors to kill PKH67 + A431 tumor cells in the presence of cetuximab (IgG) or IgA anti-EGFR (IgA) Abs at a 2:1 E:T ratio in a 12-hour FACS-based assay. One-way ANOVA with Tukey multiple comparisons tests. Summary graphs represent the total tumoricidal activity of effectors calculated as described in Materials and Methods. F–H, Representative images of GFP + A431 tumor cells cocultured with blood monocytes (patient LC441) at different E:T ratios and cetuximab for 48 hours in the IncuCyte Live Cell Analysis System. Image of A431+Ab, where Ab is a cetuximab, is intentionally the same in both and . Scale bar, 400 μm. Representative experiments showing the kinetics of A431 tumor cell growth when cocultured with blood monocytes ( F ), PBNs ( G ), and TAMs ( H ) in the presence of anti-EGFR Abs at the indicated E:T ratios in the IncuCyte Live Cell Analysis System. I and J, Summary results showing the kinetics of GFP + A431 tumor cell growth when cocultured with FcR + effectors in the presence of cetuximab or IgA anti-EGFR Ab at 2:1 E:T ratio in the IncuCyte Live Cell System. The percentage of tumor cell growth inhibition/stimulation was calculated at 48 hours. One-way ANOVA with Tukey multiple comparisons tests. K, Representative dot plots and cumulative flow cytometry data demonstrating the expression of EGFR on the surface of EpCam + cells in tumor and distant lung tissue. Paired t test. MFI, mean fluorescence intensity. L, Representative flow cytometry histograms showing the expression of EGFR on the surface of EpCam + cells in tumor and distant lung tissue in comparison with A431 and A549 tumor cells. Number of patients is indicated on the graphs. All data represented as mean ± SEM. FcR + effectors were freshly isolated for all experiments.

Article Snippet: Tumor-associated neutrophils (TAN) were isolated from tumor single-cell suspensions using positive selection with CD15 or CD66b antibody–conjugated magnetic microbeads (130–046–601 and 130–111–552, Miltenyi Biotec Inc.) according to the manufacturer's protocol as previously described ( ).

Techniques: Flow Cytometry, Activity Assay, Control, Cell Analysis, Inhibition, Expressing, Fluorescence, Comparison, Isolation