Journal: Nature microbiology

Article Title: The targets of acute norovirus infection are immune cells in the gut-associated lymphoid tissue

doi: 10.1038/s41564-017-0057-7

Figure Lengend Snippet: a–b) Duplicate wells of the indicated cell lines were infected with mock inoculum or MNV-1 at MOI 5. Virus titers were determined by TCID 50 assay on culture supernatants (a) and cell viability was assessed by propidium iodide staining (b) at the indicated time points. Error bars denote SEM. c) The frequency and abundance of cells expressing the MNV-1 receptor CD300lf were determined on naïve cells by flow cytometric staining using a matched isotype antibody as a negative control. d) Peyer’s patches cells from B6 mice (n=6) were stained with two panels of antibodies (see Methods). The frequency and abundance of DC/Mφ (CD45 + CD11c + ), B cells (CD45 + CD19 + B220 + ), T cells (CD45 + CD3 + ), and intestinal epithelial cells (CD45 − EpCAM + ) expressing CD300lf were determined. For all panels, experiments were performed three times.

Article Snippet: The following antibodies were used in one multicolor panel alongside CD300lf (panel 1): anti-mouse CD19 PE (clone 1D3, eBioscience, 12-0193), anti-mouse B220 PE-Cy7 (clone RA3-6B2, BioLegend, 103221), anti-mouse CD11c FITC (clone N418, BioLegend, 117305), and anti-mouse CD45 Pacific Blue (clone 30-F11, BioLegend, 103125).

Techniques: Infection, Virus, Staining, Expressing, Negative Control

Journal: Scientific Reports

Article Title: Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance

doi: 10.1038/s41598-017-05212-4

Figure Lengend Snippet: Increased mucosal transport of secretory antibodies in inflamed lungs. ( a ) anti-pIgR and anti-Gapdh immunoblot of 20 µg protein from whole lung homogenates of SPC-HA (n = 3) and SPC-HAxTCR-HA (n = 3) mice. Densitometric quantification of protein bands is stated in arbitrary units above each lane. Relative pIgR quantity was calculated normalizing densitometric pIgR value to the corresponding Gapdh value and subsequently comparing normalized pIgR values of the SPC-HAxTCR-HA group to the SPC-HA group. Data are representative for at least two individual experiments with similar results. ( b ) Lung tissue sections were stained with anti-pIgR (green), representative alveolar structures from n = 3/group are depicted. White circles illustrate representative densitometrically quantified tissue areas. Calculated total cell fluorescence (CTCF) was determined as: Integrated density of fluorescence-positive cell – (Area of fluorescence-positive cell × mean fluorescence intensity of background signal). Median CTCF of quantified areas in representative images are depicted as white numbers. IgA and IgM levels in ( c ) bronchoalveolar lavage fluid (BALF) and ( d ) serum of SPC-HA and SPC-HAxTCR-HA mice were determined by ELISA. ( e ) Relative secretory IgA concentrations in serial dilutions of BALF samples were determined by ELISA. Results are expressed as the mean optical density (OD) at 450 nm ± SEM, *p < 0.05, ** p < 0.01 (n = 6–7/group).

Article Snippet: Bacteria were washed once with PBS and stained using anti-mouse IgA (clone: C10-3) or anti-mouse IgM (clone: DS-1, both BD Pharmingen).

Techniques: Western Blot, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance

doi: 10.1038/s41598-017-05212-4

Figure Lengend Snippet: Increased pneumococcal binding capacities by lung mucosal fluid in inflamed lungs. Pneumococci were co-incubated with bronchoalveolar lavage fluid (BALF) supernatants from SPC-HA and SPC-HAxTCR-HA mice. Bacteria were stained with anti-mouse IgA or anti-IgM antibodies and analyzed by flow cytometry (FACS). ( a ) Representative FACS plots of IgA+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgA without prior incubation with BALF. ( b ) Percentages of IgA+ pneumococci and relative fluorescence intensities ( c ) of IgA+ pneumococci. ( d ) Representative FACS plots of IgM+ pneumococci incubated with BALF from SPC-HA or SPC-HAxTCR-mice; control samples (CTRL) were stained with anti-IgM without prior incubation with BALF. ( e ) Percentages of IgM+ pneumococci and relative fluorescence intensities ( f ) of IgM+ pneumococci. Relative fluorescence intensities are calculated by the ratio of the MFI of each individual sample over the mean MFI of the SPC-HA control group. Data are pooled from 2 independent experiments with similar results. *p < 0.05 **p < 0.01, ***p < 0.001.

Article Snippet: Bacteria were washed once with PBS and stained using anti-mouse IgA (clone: C10-3) or anti-mouse IgM (clone: DS-1, both BD Pharmingen).

Techniques: Binding Assay, Incubation, Staining, Flow Cytometry, Fluorescence