9 research outputs found

    Flow cytometry analysis of freshly isolated AEC from mouse lungs.

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    <p>Total lung cells were obtained by using Corti's protocol with some modifications as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032125#s4" target="_blank">Material and Methods</a>. Leukocytes were first depleted with anti-CD45 microbeads and subsequently, the CD45<b><sup>−</sup></b> cells were depleted of contaminating endothelial cells using anti-CD146 microbeads. The remaining CD45<sup>−</sup>CD146<sup>−</sup> cells were considered to be AEC. These cells were fixed and stained intracellularly with antibodies to CD74 (AEC II marker) and podoplanin (T1α) (AEC I marker). A representative dot plot of flow cytometry analysis of CD74 and T1α expression in freshly isolated AEC from three independent experiments is shown. Percentage numbers represent gated cells from total cells.</p

    Quantitative measurements of selected factors in supernatants from AEC.

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    <p>AEC (5×10<sup>4</sup> cells per well) were stimulated with the indicated stimuli for 24 h. IL-6, KC, GM-CSF, RANTES, MCP-1, and IP-10 levels were measured in cell culture supernatants using ELISA. Values are expressed as means ± SD, from 3 independent experiments. * represents levels significantly different from unstimulated control, p<0.05. The dotted line indicates the concentration in supernatants from unstimulated cells.</p

    Uptake of mycobacteria by AEC and PuM.

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    <p>Isolated AEC (A and B) and PuM (C and D) were cultured on cover glass at a concentration of 1×10<sup>5</sup> cells per well as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032125#s4" target="_blank">Material and Methods</a>. After overnight culture in medium without antibiotics, the cells were infected with GFP-BCG at a MOI of 1∶100 (cell∶bacteria) for 4 h at 37°C in RPMI medium without antibiotics. To kill all extracellular remained bacteria, the cells were treated with gentamicin for 1 h, washed 3 times and finally incubated for 72 h in RPMI without antibiotics. After that, the cover glasses with infected cells were fixed, mounted and observed under white light (A and C) and green light (B and D) in a fluorescence microscope. Magnification 1000×.</p

    Mycobacterial uptake and intracellular growth by AEC and PuM.

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    <p>Data are expressed in relative luminescence units (RLU). Uptake is measured at 0 h and intracellular growth at 72 h. Increase in RLU from GFP-BCG cultured in cell free medium was at 72 h, 6 fold of the measured at 0 h. This value was used as a reference to bacterial growth. Data are an average from four different experiments and expressed as mean ± SEM, n = 15.</p

    Chemokine production by primary AEC and PuM after <i>in vitro</i> stimulation.

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    <p>Data are expressed in pg/ml of cytokine produced. A representative experiment from five independent experiments is shown. Values are given as mean ± SD n = 3–4. ND: not detected.</p

    Cytokine/chemokine profiling on supernatants from AEC.

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    <p>AEC (5×10<sup>4</sup> cells per well) were stimulated with the indicated stimuli for 24 h. Supernatants were incubated on membranes from a mouse Cytokine Array Panel A Proteome Profiler kit (R&D systems) according to the manufacturer's instructions. The figure shows a+representing a spot with similar or greater density than control spots, whereas +/− indicates a spot with lower density than control spots.</p

    Chemokine production by the cell lines T7 and MH-S after <i>in vitro</i> stimulation.

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    <p>Data are expressed in pg/ml of cytokine produced. A representative experiment from five independent experiments is shown. Values are given as mean ± SD n = 3–4. ND: not detected.</p

    Antigen presentation by AEC.

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    <p>IFN-γ levels after <i>in vitro</i> restimulation of splenocytes from Ag19 kDa immunized mice. As APC we used AEC (AEC<sub>19 kDa</sub>) and PuM (PuM<sub>19 kDa</sub>) pulsed with Ag19 kDa as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032125#s4" target="_blank">Materials and Methods</a>. One week after the last immunization with Ag19 kDa, mice were sacrificed and splenocytes from immunized and unimmunized mice were co-cultured with AEC<sub>19 kDa</sub> and PuM<sub>19 kDa</sub> for 72 h. Data are expressed as mean ± SD from 3 mice per group. A representative of two independent experiments is shown. * represents levels significantly different from unimmunized control, p<0.05.</p

    MMP-9 production by primary cells.

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    <p>Data are expressed in pg/ml of cytokine produced by primary cells. A representative experiment from five independent experiments is shown. Values are given as mean ± SD n = 3–4. ND: not detected.</p
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