Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: C15 inoculation of airway epithelial cells causes a speckled pattern of infection and infected cell shedding. HBEC-ALI cultures were inoculated for 18 h with C15 or media alone and imaged by fluorescent microscopy ( a and b , respectively). Nuclei stained with Hoechst ( blue ), C15 capsid stained with monoclonal antibody against VP2 ( red ). Inoculated cultures were also imaged by confocal microscopy and analyzed by z-stacking ( c and d ) or apical surface views ( e and f ). Nuclei stained with Syto-13 ( green ), secretory cells stained with WGA ( blue ), and C15 capsid stained with monoclonal antibody ( orange ). Scale bars indicate 20 μm

Article Snippet: Slides were blocked (3% FBS, 2% goat serum, 0.2% Tween-20, 1.25% Human BD Fc BlockTM, 1 h, RT), incubated (1:200 in blocking buffer, 2 h, RT) with anti-C15-VP2 mouse monoclonal antibody (kindly provided by MedImmune Inc., Gaithersberg MD), Mach 4 Universal Probe and then Polymer (15 min, RT each, Biocare Medical, Concord, CA), Betazoid DAB (5 min, RT, Biocare Medical, Concord, CA), and counterstained with CAT hematoxylin or eosin (30s, RT, Biocare Medical, Concord, CA).

Techniques: Infection, Microscopy, Staining, Confocal Microscopy

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Analysis of C15 infectivity by immunofluorescent staining. HBEC-ALI cultures were infected for 18 h with C15 and imaged apically and z-stacked orthogonally by confocal microscopy. Secretory and ciliated cells were stained with WGA and rabbit polyclonal antibody against aat, respectively ( false color red ), C15 capsid stained with monoclonal antibody ( false color green ), nuclei stained with Syto-13 ( false color blue ). Scale bars indicate 20 μm

Article Snippet: Slides were blocked (3% FBS, 2% goat serum, 0.2% Tween-20, 1.25% Human BD Fc BlockTM, 1 h, RT), incubated (1:200 in blocking buffer, 2 h, RT) with anti-C15-VP2 mouse monoclonal antibody (kindly provided by MedImmune Inc., Gaithersberg MD), Mach 4 Universal Probe and then Polymer (15 min, RT each, Biocare Medical, Concord, CA), Betazoid DAB (5 min, RT, Biocare Medical, Concord, CA), and counterstained with CAT hematoxylin or eosin (30s, RT, Biocare Medical, Concord, CA).

Techniques: Infection, Staining, Confocal Microscopy

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Immunohistochemical analysis of C15 infectivity in HBEC-ALI cultures. PCM-differentiated HBEC-ALI cultures were inoculated with a C15 or b media alone, stained for C15 capsid, and imaged by light microscopy. C15 capsid is represented by brown-staining cells . Scale bars indicate 20 μm

Article Snippet: Slides were blocked (3% FBS, 2% goat serum, 0.2% Tween-20, 1.25% Human BD Fc BlockTM, 1 h, RT), incubated (1:200 in blocking buffer, 2 h, RT) with anti-C15-VP2 mouse monoclonal antibody (kindly provided by MedImmune Inc., Gaithersberg MD), Mach 4 Universal Probe and then Polymer (15 min, RT each, Biocare Medical, Concord, CA), Betazoid DAB (5 min, RT, Biocare Medical, Concord, CA), and counterstained with CAT hematoxylin or eosin (30s, RT, Biocare Medical, Concord, CA).

Techniques: Immunohistochemical staining, Infection, Staining, Light Microscopy

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Single cell analysis of RV-C infection and epithelial cell surface markers. HBEC-ALI cultures infected or mock-infected for 18 h with C15 or BEGM alone, respectively, were labeled with antibodies against C15 capsid (C15), and either a acetylated-alpha-tubulin (aat, Cilia) or b fluorescently labeled-WGA (WGA) and analyzed by flow cytometry. The graphs summarize the percentage of C15+ cells in ciliated and non-ciliated cells ( n = 4 independent experiments, two cell donors). Mock, mock-inoculated cultures; C15, C15-inoculated cultures. *** p < 0.001

Article Snippet: Slides were blocked (3% FBS, 2% goat serum, 0.2% Tween-20, 1.25% Human BD Fc BlockTM, 1 h, RT), incubated (1:200 in blocking buffer, 2 h, RT) with anti-C15-VP2 mouse monoclonal antibody (kindly provided by MedImmune Inc., Gaithersberg MD), Mach 4 Universal Probe and then Polymer (15 min, RT each, Biocare Medical, Concord, CA), Betazoid DAB (5 min, RT, Biocare Medical, Concord, CA), and counterstained with CAT hematoxylin or eosin (30s, RT, Biocare Medical, Concord, CA).

Techniques: Single-cell Analysis, Infection, Labeling, Flow Cytometry

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Highly differentiated cells are more susceptible to RV-C infection. Differentiated cultures were incubated for 18 h after inoculation with C15 or BEGM alone, labeled with antibodies against C15 capsid (C15) and either a aat (Cilia) or b Muc5AC and analyzed by flow cytometry. The graphs show the percentage of C15-positive cells by cell type ( n = 6). *** p < 0.001

Article Snippet: Slides were blocked (3% FBS, 2% goat serum, 0.2% Tween-20, 1.25% Human BD Fc BlockTM, 1 h, RT), incubated (1:200 in blocking buffer, 2 h, RT) with anti-C15-VP2 mouse monoclonal antibody (kindly provided by MedImmune Inc., Gaithersberg MD), Mach 4 Universal Probe and then Polymer (15 min, RT each, Biocare Medical, Concord, CA), Betazoid DAB (5 min, RT, Biocare Medical, Concord, CA), and counterstained with CAT hematoxylin or eosin (30s, RT, Biocare Medical, Concord, CA).

Techniques: Infection, Incubation, Labeling, Flow Cytometry

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: CDHR3 is predominantly expressed by ciliated epithelial cells and diminishes following C15 inoculation. PC-differentiated cultures inoculated or mock-inoculated for 18 h with C15 (C15+) or BEGM alone (Mock), respectively, were labeled with antibodies against C15 capsid (C15), CDHR3, and aat (cilia) and analyzed by flow cytometry ( n = 6). a Relative median fluorescence intensity (rMFI) of CDHR3 in ciliated (Yes) and nonciliated (No) cell populations of mock-inoculated cultures. b Frequency of CDHR3 positive ciliated cells out of the total ciliated cell population in mock- and C15-inoculated cultures. c rMFI of CDHR3 in ciliated cells negative (C15-) or positive (C15+) for C15 staining. Values are normalized to the double-negative (nonciliated and CDHR3-) population in each experiment. ** p ≤ 0.01, * p ≤ 0.05

Article Snippet: Slides were blocked (3% FBS, 2% goat serum, 0.2% Tween-20, 1.25% Human BD Fc BlockTM, 1 h, RT), incubated (1:200 in blocking buffer, 2 h, RT) with anti-C15-VP2 mouse monoclonal antibody (kindly provided by MedImmune Inc., Gaithersberg MD), Mach 4 Universal Probe and then Polymer (15 min, RT each, Biocare Medical, Concord, CA), Betazoid DAB (5 min, RT, Biocare Medical, Concord, CA), and counterstained with CAT hematoxylin or eosin (30s, RT, Biocare Medical, Concord, CA).

Techniques: Labeling, Flow Cytometry, Fluorescence, Staining

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: CDHR3 expression in differentiating bronchial epithelial cells positively correlates with RV-C binding and replication. a CDHR3 protein expression (~100 kDa) and b mRNA expression ( n = 8) by differentiating airway epithelial cells in ALI using PCM from day 1 to day 28. c BECs in ALI were infected weekly with C15 and the infected cell samples were collected and analyzed for viral binding (2 hpi) and viral yield (24 hpi). d and e Correlations (linear regression) between cellular CDHR3 mRNA expression and C15 binding and yield

Article Snippet: Slides were blocked (3% FBS, 2% goat serum, 0.2% Tween-20, 1.25% Human BD Fc BlockTM, 1 h, RT), incubated (1:200 in blocking buffer, 2 h, RT) with anti-C15-VP2 mouse monoclonal antibody (kindly provided by MedImmune Inc., Gaithersberg MD), Mach 4 Universal Probe and then Polymer (15 min, RT each, Biocare Medical, Concord, CA), Betazoid DAB (5 min, RT, Biocare Medical, Concord, CA), and counterstained with CAT hematoxylin or eosin (30s, RT, Biocare Medical, Concord, CA).

Techniques: Expressing, Binding Assay, Infection