Journal: Frontiers in Microbiology

Article Title: Combining flagellin and human β-defensin-3 to combat bacterial infections

doi: 10.3389/fmicb.2014.00673

Figure Lengend Snippet: Effect of F protein on expression levels of IL12A and IFNγ in CD4+ T cells from healthy donors. (A–C) Effect of F protein on expression levels of IL12A and IFNγ in naïve and activated CD4+ T cells. (A) Experimental design for naïve cells – CD4+ T cells were isolated from peripheral blood of healthy donors using CD4+ magnetic-beads. CD4+ T cells were seeded and incubated with the F protein (2 μg/ml) for 18 or 24 h. (B) Experimental design for activated cells – mononuclear cells were activated by PHA (20 μg/ml) for 72 h and then grown with IL2 (10 units/ml recombinant IL-2). Following activation, CD4+ T cells were separated using CD4 magnetic beads. CD4+ T cells were seeded and incubated with the F protein (2 μg/ml) for 24 or 48 h. Following the incubation, cDNA samples were prepared and subjected to real-time-PCR analysis using SYBR green reagent (as described in Methods). (C) Changes in IFNγ-mRNA and IL12A-mRNA levels upon F stimuli, compared to control cells. I = one F stimulation. Data represents the mean ± SD of 3–5 separate experiments. (D,E) Effect of the F protein on expression levels of IL12A and IFNγ in naïve CD4+ T cells after triggering all mononuclear cells. (D) Experimental design – Naïve mononuclear cells were incubated with flagellin for different time periods, after which CD4+ T cells were separated using CD4 magnetic beads. The separated CD4+ T cells were taken and cDNA samples were prepared and subjected to real time-PCR analysis using SYBR green reagent (as described in Methods). (E) The changes of IFNγ-mRNA and IL12A-mRNA in CD4+ T cells levels upon flagellin stimuli in the presence of mononuclear cells after 2, 6, 12, 24, and 36 h. I = one F stimulation. Data represents the mean ± SD of 3–5 separate experiments. (F,G) Effect of the F protein on expression levels of IL12A and IFNγ in naïve CD4+ T cells after triggering the mononuclear cells three times. (F) Experimental design – Naïve mononuclear cells were triggered three times with flagellin for 2 or 12 h stimuli. Following stimulation, CD4+ T cells were separated using cd4 magnetic beads. The separated CD4+ T cells were removed and cDNA samples were prepared and subjected to real time PCR analysis using SYBR green reagent (as described in Methods). (G) Changes of IFNγ- and IL12A mRNA levels upon repeated flagellin stimulations for 2 or 12 h each. III = three F stimulations. Data represents the mean ± SD of 3–5 separate experiments.

Article Snippet: For the CD4+ T cells additional staining was performed with anti-human CD4-PE-conjugated (1:50; Serotec, Oxford, UK).

Techniques: Expressing, Isolation, Magnetic Beads, Incubation, Recombinant, Activation Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Frontiers in Microbiology

Article Title: Combining flagellin and human β-defensin-3 to combat bacterial infections

doi: 10.3389/fmicb.2014.00673

Figure Lengend Snippet: Effect of the F protein on expression levels of TLR5 in healthy CD4+ T cells. (A,B) Changes of TLR5-mRNA levels in naïve (A) or activated (B) CD4+ T cells upon one F stimulation after 18 or 24 h. (C,D) Changes of TLR5-mRNA levels following repeated (three) F stimulations in naïve (C) and activated (D) CD4+ T cells. (E) Changes of TLR5-mRNA levels following one F stimulation after various incubation times. Data for each graph represents the mean ± SD of 3–5 separate experiments (A,B,E) or one representative experiment out of two performed is shown (C,D) .

Article Snippet: For the CD4+ T cells additional staining was performed with anti-human CD4-PE-conjugated (1:50; Serotec, Oxford, UK).

Techniques: Expressing, Incubation

Journal: Cell reports

Article Title: Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment

doi: 10.1016/j.celrep.2022.111201

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For subsequent analysis by flow cytometry, tumors were cut into pieces and digested with either Collagenase Ia (1mg/mL) C2674 Sigma Aldrich and Hyaluronidase V (0.1mg/mL) H6254 Sigma Aldrich for 40min at 37°C or with a mouse tumor dissociation kit (Miltenyi Biotec #130-096-730) using gentle MACS dissociator.

Techniques: Control, Flow Cytometry, Virus, shRNA, Recombinant, Plasmid Preparation, Western Blot, Electron Microscopy, Activation Assay, DNA Purification, SYBR Green Assay, Reverse Transcription, Endotoxin Assay, Software, Cell Counting, Staining, Membrane

Journal: PLoS Medicine

Article Title: Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration

doi: 10.1371/journal.pmed.0030310

Figure Lengend Snippet: (A–C) On day 7 following laser treatment, whole mount stains were performed to determine the cells present in the area of the neovascular complex. Stains for CD11b were performed on (A) B6, (B) IL-10 −/− , and (C) B6 mice treated with neutralizing anti-IL-10. Images were take by confocal microscopy (magnification 200×) centered on the laser lesion. (D) The number of CD11b + cells per lesion was counted (200× high-power field centered on the laser lesion) (B6, 6.3 ± 0.9; IL10 −/− , 22.6 ± 2.1; B6 + anti-IL10, 22.5 ± 2.5). Asterisks indicate values significantly different from control. (E) Dual staining was performed on day 7 following laser treatment using FITC-conjugated anti-CD11b (green) and PE-conjugated anti-F4/80 (red) (magnification 400×).

Article Snippet: Anti-CD11b (5C6), anti-F4/80 (C1;A3–1), and the isotype control (IgG2b) were purchased from Serotec (Raleigh, North Carolina, United States).

Techniques: Confocal Microscopy, Staining

Journal: PLoS Medicine

Article Title: Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration

doi: 10.1371/journal.pmed.0030310

Figure Lengend Snippet: (A) IL-10 −/− mice were injected with anti-CD11b (volume of the neovascular complex: 12,903.1 ± 2,171.3 μm 3 ), anti-F4/80 (9,977.3 ± 1,572.7 μm 3 ), or control IgG (2,262.3 ± 313.4 μm 3 ) on days −1, 0, and +1. CNV volumes were determined on day 7. (B) IL-10 −/− mice were injected in the vitreous with PBS (1,394 ± 382.4 μm 3 ) or 100 ng of rIL-10 on day 0 (the day of laser treatment; 4,033.6 ± 1,026.5 μm 3 ) or day 3 (6,949.8 ± 1,475.5 μm 3 ). (C and D) Cross-sections of the retinas of a control littermate (C) and a VMD2-IL-10 Tg mouse (D) stained with anti-IL-10 and examined by confocal microscopy. (E) Hematoxylin and eosin staining of the retina of a VMD2-IL-10 Tg mouse (magnification 200×). (F) VMD2-IL-10 Tg mice (24,428.6 ± 4,360.7 μm 3 ) and littermate controls (6,830.1 ± 1,324.9 μm 3 ) were subjected to laser treatment and CNV volumes were determined on day 7. Asterisks indicate values significantly different from control; p- values are given in parentheses.

Article Snippet: Anti-CD11b (5C6), anti-F4/80 (C1;A3–1), and the isotype control (IgG2b) were purchased from Serotec (Raleigh, North Carolina, United States).

Techniques: Injection, Staining, Confocal Microscopy

Journal: PLoS Medicine

Article Title: Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration

doi: 10.1371/journal.pmed.0030310

Figure Lengend Snippet: Cells obtained from bone marrow (BM) cultures (A) or spleen (B) were purified by magnetic beads and injected into the vitreous cavity on the day of laser treatment. Seven days later, the volume of the neovascular complex was determined by confocal microscopy. Asterisks indicate values significantly different from control; p- values given in parentheses are based on Student's t test. (A) Volume of the neovascular complex for injection of PBS (14,470.9 ± 1,636.4 μm 3 ), CD11b cells—1 × 10 5 cells (2,431.2 ± 551.3 μm 3 ), and CD11b cells—5 × 10 5 cells (5,274.2 ± 772.5 μm 3 ). (B) Volume of the neovascular complex for injection of PBS (12,857.9 ± 1,094.9 μm 3 ), CD3 cells (14,960.5 ± 2,502.0 μm 3 ), CD11b cells (4,135.1 ± 714.4 μm 3 ), and CD11c cells (15,063.3 ± 1,982.4 μm 3 ).

Article Snippet: Anti-CD11b (5C6), anti-F4/80 (C1;A3–1), and the isotype control (IgG2b) were purchased from Serotec (Raleigh, North Carolina, United States).

Techniques: Purification, Magnetic Beads, Injection, Confocal Microscopy

Journal: PLoS Medicine

Article Title: Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration

doi: 10.1371/journal.pmed.0030310

Figure Lengend Snippet: (A) CD11b + cells from B6, B6- lpr, and B6- gld mice were purified from spleen, and 1 × 10 5 cells were injected into the vitreous cavity on the day of laser induction of CNV. Seven days later, the volume of the neovascular complex was determined by confocal microscopy for PBS (11,786.4 ± 1,907.3 μm 3 ), B6 (2,084.6 ± 874.8 μm 3 ), gld (15,824.9 ± 1,483.6 μm 3 ), and lpr (5,443.9 ± 542.1 μm 3 ). Asterisks indicate values significantly different from control. (B) Purified CD11b + cells were cultured overnight with LPS or necrotic retina and tested for killing against L1210-Fas. (C) The expression of CD95L in CD11b cells was determined by flow cytometry following overnight culture with LPS (dotted line) or necrotic retina (solid line). The line with shading underneath represents untreated cells.

Article Snippet: Anti-CD11b (5C6), anti-F4/80 (C1;A3–1), and the isotype control (IgG2b) were purchased from Serotec (Raleigh, North Carolina, United States).

Techniques: Purification, Injection, Confocal Microscopy, Cell Culture, Expressing, Flow Cytometry