Journal: PLoS ONE

Article Title: Alpha-1 antitrypsin supplementation improves alveolar macrophages efferocytosis and phagocytosis following cigarette smoke exposure

doi: 10.1371/journal.pone.0176073

Figure Lengend Snippet: A . Absolute efferocytosis index representing % of cells that engulfed fluorescently labeled apoptotic targets among AM isolated from active smokers (dark circles) compared to those from healthy non-smokers (light gray circles). Native A1AT (Aralast NP, 100 μg/mL, 16 h) significantly increased efferocytosis in AM from smokers (dark gray circles), but not in those from healthy non-smokers (gray squares). 1-way ANOVA, Sidak’s multiple comparisons test, * p<0.05. B-C . Relative efferocytosis index representing % of AM that engulfed fluorescently labeled apoptotic targets following indicated exposures, when compared to those exposed to control media. Primary rat AM ( B ) or NR8383 AM ( C ) were exposed ex-vivo to AC or CS (4h, 3% or 10%, respectively, 4 h), native A1AT (Aralast NP, 100 μg/mL, 16 h), and compared to IL-4 (20 ng/mL, 72 h). D . Polymerized A1AT (Aralast NP, 100 μg/mL, 16h) has no effect on CS-exposed NR8383 AM efferocytosis. Data are presented as mean ± SEM, 1-way ANOVA, Sidak’s multiple comparisons test, * p<0.05, ** p<0.001, *** p<0.0001.

Article Snippet: Phorbol 12-myristate 13-acetate (PMA), rabbit anti-MAR antibody, rabbit anti-iNOS antibody, rabbit polyclonal anti-GAPDH, mouse monoclonal anti-vinculin were from Abcam (Cambridge, MA, USA), rabbit anti-SRB-2 antibody was from Santa Cruz Biotechnology (Dallas, TX, USA), mouse anti-human CD3 was from BD Biosciences (Franklin Lakes, NJ, USA), recombinant rat IL-4 was from ProSpec-Tany TechnoGene (Ness-Ziona, Israel), and TNF-α Protease Inhibitor-1 (TAPI- 1 ) was from ESD Millipore (Billerica, MA, USA).

Techniques: Labeling, Isolation, Ex Vivo

Journal: Frontiers in Immunology

Article Title: High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

doi: 10.3389/fimmu.2021.611280

Figure Lengend Snippet: NR8383 rat macrophage activation status after stimulation with (A) LPS and IFN-γ, (B) IL-4 or (C) IL-4 or IL-4/IL-13. Data are normalized to cell count and presented as mean values ± SEM of three separate experiments. Statistical comparisons are made using a one-way ANOVA analysis with Bonferroni’s multiple comparison post-hoc test. Results are compared to non-polarized control (M0) and statistical significance is marked as follows: * indicates p < 0.01. (C) Representative fluorescent dot (quadrants) plots showing percentage of cells expressing MRC-1 surface receptor.

Article Snippet: Lipopolysaccharide (LPS, cat#L2630, Sigma Aldrich, Dorset, UK), recombinant rat interferon gamma (IFN-γ, cat#550072), and recombinant rat interleukin 4 (IL-4, cat#555107) were purchased from BD Biosciences, (Wokingham, Berkshire, UK), while recombinant rat interleukin 13 (IL-13, cat#CB243IL13) was purchased from PAN-Biotech (Wimborne, Dorset, UK).

Techniques: Activation Assay, Cell Counting, Expressing

Journal: Frontiers in Immunology

Article Title: High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

doi: 10.3389/fimmu.2021.611280

Figure Lengend Snippet: Morphological characteristic of non-activated, M(LPS+/IFN-γ+) or M(IL-4+) activated NR8383 rat macrophages. Nuclear area (A) , cellular area (B) , number vacuoles per cell (C) , average area of a single vacuole (D) , cellular area occupied by all vacuoles (E) and total area of all vacuoles per cell (F) were quantified in non-activated macrophages (M0), activated with 100 ng/mL LPS and 20 ng/mL IFN-γ [M(LPS+/IFN-γ+)] or activated with 30 ng/mL IL-4 [M(IL-4+)]. Data are presented as mean values ± SEM of three separate experiments. Statistical comparisons are made using a one-way ANOVA analysis with Bonferroni’s multiple comparison post-hoc test. Results are compared to non-polarized control (M0); *** indicates p < 0.001.

Article Snippet: Lipopolysaccharide (LPS, cat#L2630, Sigma Aldrich, Dorset, UK), recombinant rat interferon gamma (IFN-γ, cat#550072), and recombinant rat interleukin 4 (IL-4, cat#555107) were purchased from BD Biosciences, (Wokingham, Berkshire, UK), while recombinant rat interleukin 13 (IL-13, cat#CB243IL13) was purchased from PAN-Biotech (Wimborne, Dorset, UK).

Techniques:

Journal: Frontiers in Immunology

Article Title: High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

doi: 10.3389/fimmu.2021.611280

Figure Lengend Snippet: Functional characteristic of non-activated, M(LPS+/IFN-γ+) or M(IL-4+) activated NR8383 rat macrophages. Cell count (A) , phagocytic activity (B) , cellular phospholipid (C) and cellular neutral lipid content (D) were quantified in non-activated macrophages (M0), activated with 100 ng/ml LPS and 20 ng/ml IFN-γ [M(LPS+/IFN-γ+)] or activated with 30 ng/ml IL-4 [M(IL-4+)]. Data are normalized to cell count and presented as mean values ± SEM of three separate experiments. Statistical comparisons are made using a one-way ANOVA analysis with Bonferroni’s multiple comparison post-hoc test. Results are compared to non-polarized control (M0); ** indicate p < 0.01, *** indicate p < 0.001.

Article Snippet: Lipopolysaccharide (LPS, cat#L2630, Sigma Aldrich, Dorset, UK), recombinant rat interferon gamma (IFN-γ, cat#550072), and recombinant rat interleukin 4 (IL-4, cat#555107) were purchased from BD Biosciences, (Wokingham, Berkshire, UK), while recombinant rat interleukin 13 (IL-13, cat#CB243IL13) was purchased from PAN-Biotech (Wimborne, Dorset, UK).

Techniques: Functional Assay, Cell Counting, Activity Assay

Journal: Frontiers in Immunology

Article Title: High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

doi: 10.3389/fimmu.2021.611280

Figure Lengend Snippet: Effect on arginase activity and MRC-1 receptor expression in M0-non-activated and IL-4 activated NR8383 rat macrophages after 24 h exposure to 5 µM amiodarone and 0.1 µM staurosporine. (A) Effect on arginase activity. Data are normalized to cell count and presented as mean values ± SEM of three separate experiments. Statistical comparisons are made using a one-way ANOVA analysis with Bonferroni’s multiple comparison post-hoc test. Results are compared to non-polarized control (M0); *** indicates p < 0.001. (B) Representative fluorescent dot (quadrants) plots showing percentage of cells expressing MRC-1 surface receptor.

Article Snippet: Lipopolysaccharide (LPS, cat#L2630, Sigma Aldrich, Dorset, UK), recombinant rat interferon gamma (IFN-γ, cat#550072), and recombinant rat interleukin 4 (IL-4, cat#555107) were purchased from BD Biosciences, (Wokingham, Berkshire, UK), while recombinant rat interleukin 13 (IL-13, cat#CB243IL13) was purchased from PAN-Biotech (Wimborne, Dorset, UK).

Techniques: Activity Assay, Expressing, Cell Counting

Journal: Frontiers in Immunology

Article Title: High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

doi: 10.3389/fimmu.2021.611280

Figure Lengend Snippet: Phagocytic activity in M0-non-activated, LPS/IFN-γ or IL-4 activated NR8383 rat macrophages after 24 h exposure to 5 µM amiodarone (orange bars) and 0.1 µM staurosporine (purple bars). Data are expressed as a percentage of the phagocytic activity of non-polarized untreated NR8383 cells. Data represent the mean values ± SEM of three separate experiments. Statistical comparisons are made using a one-way ANOVA analysis with Bonferroni’s multiple comparison post-hoc test. Results are compared to non-polarized control (M0); ** indicate p < 0.01, *** indicates p < 0.001.

Article Snippet: Lipopolysaccharide (LPS, cat#L2630, Sigma Aldrich, Dorset, UK), recombinant rat interferon gamma (IFN-γ, cat#550072), and recombinant rat interleukin 4 (IL-4, cat#555107) were purchased from BD Biosciences, (Wokingham, Berkshire, UK), while recombinant rat interleukin 13 (IL-13, cat#CB243IL13) was purchased from PAN-Biotech (Wimborne, Dorset, UK).

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

doi: 10.3389/fimmu.2021.611280

Figure Lengend Snippet: Phospholipid (A) and neutral lipid (B) content in M0-non-activated, LPS/IFN-γ and IL-4 activated NR8383 rat macrophages after 24 h exposure to 5 µM amiodarone (orange bars) and 0.1 µM staurosporine (purple bars). Data are normalized to cell count and presented as mean values ± SEM of three separate experiments. Statistical comparisons are made using a one-way ANOVA analysis with Bonferroni’s multiple comparison post-hoc test. Results are compared to non-polarized control (M0); *** indicates p < 0.001.

Article Snippet: Lipopolysaccharide (LPS, cat#L2630, Sigma Aldrich, Dorset, UK), recombinant rat interferon gamma (IFN-γ, cat#550072), and recombinant rat interleukin 4 (IL-4, cat#555107) were purchased from BD Biosciences, (Wokingham, Berkshire, UK), while recombinant rat interleukin 13 (IL-13, cat#CB243IL13) was purchased from PAN-Biotech (Wimborne, Dorset, UK).

Techniques: Cell Counting