anti-muc5ac af700 clone45m1  (Novus Biologicals)

Journal: ERJ Open Research

Article Title: Ciliated epithelial cell differentiation at air–liquid interface and respiratory syncytial virus infection using animal-free media and substrates

doi: 10.1183/23120541.00028-2025

Figure Lengend Snippet: Barrier integrity and ciliated epithelial cell differentiation of human bronchial epithelial cells (BECs) at air–liquid interface (ALI). a,b) Epithelial integrity of BECs differentiated from one donor at ALI on membrane inserts coated with human placenta collagen IV for 4 weeks (n=8) compared with our non-animal-free (AF)model (n=10) that contains an additional Matrigel (mouse-derived) layer. Barrier integrity was measured by trans-epithelial electrical resistance (TEER) (Ω·cm −2 ) (a) and dextran permeability (μg·mL −1 ) (b). Error bars represent mean± sem of n=8–10 independent Transwells. *: p<0.05 (unpaired ANOVA with Benjamini–Hochberg correction for multiple comparisons). c) Histogram of ciliary beat frequency (CBF) (Hz) was quantified via high-speed video microscopy of 6400 regions of interest (ROIs) from five independent areas (one airway epithelial cell (AEC) donor). The red dotted line represents the mean CBF value of all areas. d,e) Representative brightfield image (d) and activity map (e) of one area (field of view) to show the position and distribution of motile ciliated cells. Scale bar, 50 μm. f) Example flow cytometry gating strategy to determine the population of basal, ciliated and goblet cells in inverted ALI cultures. Basal cells were defined as the proportion of CD49f/CD127 double-positive cells in the live cell population. Ciliated cells were then gated from the nonbasal cell population based on acetyl-α-tubulin + and CD66c − expression, whereas goblet cells were further separated from the nonciliated population based on positive expression for MUC5AC. g) Proportion (%) of basal (CD49f + CD271 + ), ciliated (acetyl-α-tubulin + CD66c − ) and goblet (MUC5AC + ) cells from one AEC donor (n=3 wells). Red dot and error bars represent mean± sem of n=3 wells.

Article Snippet: MUC5AC , Goblet cells , PE , Miltenyi Biotec (130-127-552) , No , 1:50.

Techniques: Cell Differentiation, Membrane, Derivative Assay, Permeability, Microscopy, Activity Assay, Flow Cytometry, Expressing

Journal: Journal of Virology

Article Title: Air-liquid interface model for influenza aerosol exposure in vitro

doi: 10.1128/jvi.00619-25

Figure Lengend Snippet: Characterization and FLUAV replication in differentiated BCi-NS1.1 cells. BCi-NS1.1 cells were cultured under ALI conditions for 17 days. ( A ) Paraffin-embedded cross sections of BCi-NS1.1 cells prior to ALI conditions (left) and after 17 days in ALI (right) with H&E staining. Images were captured at 40× magnification. ( B ) Paraffin-embedded cross sections of ALI cultured BCi-NS1.1 cells stained by immunofluorescence with KRT5 (basal cell marker; green), DAPI (cell nuclei; blue), and β-tubulin I and II (ciliated cell marker; red). Images were captured using a Nikon confocal microscope with NIS Elements software at 40× (left) and 60× (right). ( C ) ALI cultured BCi-NS1.1 cells were fixed with 4% paraformaldehyde and stained for β-tubulin I and II (ciliated cell marker; red), CC16 (club cell marker; red), MUC5AC (mucin marker), TFF3 (goblet cell marker), actin (green), and DAPI (cell nuclei; blue). Each panel with separate staining from the same column was merged in the last row. Images were captured using a Nikon confocal microscope with NIS Elements software at 60×. ( D ) ALI-cultured BCi-NS1.1 cells were fixed with 4% paraformaldehyde and stained for ⍺2,3- (red) and ⍺2,6- (green) SA distribution. Integrated density was calculated from fluorescent confocal images using ImageJ and divided by the number of nuclei. Statistical analysis was performed using a paired t -test and analyzed in triplicates. ( E ) ALI-cultured BCi-NS1.1 cells were infected with 1 MOI of H1N1, H3N2, and H9N2. Samples were collected from the apical part of the trans-wells at 0, 12, 24, 48, and 72 h post-infection. The samples were titrated by TCID 50 . Viruses are distinguished by differences in color. Experiments were carried out independently at least twice, each time in triplicate.

Article Snippet: The samples were then stained with the following primary antibodies: KRT5 (basal cell; 2 μg/mL; HPA059479; Millipore Sigma, MA, USA), beta-tubulin I and II (cilia; 1/800; T8535, Sigma-Aldrich, MO, USA), CC16 (Club cell; 2 μg/mL; RD181022220-01; BioVendor, NC, USA), MUC5AC (M1 mucin; 2 μg/mL; MA5-12178, ThermoFisher, MA, USA), and TFF3 (goblet cell; 2 μg/mL; HPA035464; Millipore Sigma, MA, USA) for 1 h at room temperature.

Techniques: Cell Culture, Staining, Immunofluorescence, Marker, Microscopy, Software, Infection