af488 conjugated donkey anti mouse antibody  (Biotium)

Journal: bioRxiv

Article Title: Limited response of primary nasal epithelial cells to Bordetella pertussis infection and the effector protein BteA

doi: 10.1101/2025.02.03.636266

Figure Lengend Snippet: (A) hNECs are permissive to infection. hNECs were apically infected with Bp WT at a multiplicity of infection (MOI) of 10:1. After 6 hours, the medium was discarded, and bacterial colony-forming units (CFUs) associated with hNECs per Transwell membrane were quantified immediately or after additional incubation as indicated. Data represent two independent experiments. (B) Infection with B. pertussis does not change the composition of the hNEC culture. hNECs were apically infected with Bp WT at MOI 10:1 and analyzed by flow cytometry 24 hours post-infection as outlined in the legend of . (C) B. pertussis replicates in mucus and on cilia. hNECs were infected by Bp WT expressing fluorescent mScarlet protein. ZO-1 (yellow) was stained using an anti-ZO-1 antibody, followed by an anti-rabbit IgG-DYL405 conjugate, while cilia (cyan) were labeled with an anti-acetylated tubulin antibody, followed by an anti-mouse IgG-AF488 conjugate. Bacteria were visualized by mScarlet expression (magenta). Maximum intensity projections (Z-max) from confocal Z-stack images are shown, and they are representative of two independent experiments. Scale bars, 5 μm. (D) Transepithelial electrical resistance after infection. hNECs were apically infected as above with Bp WT, Bp Δ bteA, or Bp Δ bteA::bteA at MOI 10:1. Transepithelial electrical resistance across the cell layers was measured at the indicated time points in two replicates.

Article Snippet: The next day, the Transwell membranes were washed with PBST (3x 5 min) and incubated with the anti-rabbit IgG-DyLight-405 conjugate (Jackson Immunoresearch, Cat# 111-475-003) at a dilution of 1:300, and the anti-mouse donkey IgG-AF488 conjugate (Jackson Immunoresearch, Cat# 115-546-062) at a dilution of 1:500 in 1% BSA in PBST for 1 hour at RT.

Techniques: Infection, Membrane, Incubation, Flow Cytometry, Expressing, Staining, Labeling, Bacteria

Journal: bioRxiv

Article Title: Limited response of primary nasal epithelial cells to Bordetella pertussis infection and the effector protein BteA

doi: 10.1101/2025.02.03.636266

Figure Lengend Snippet: BpWT expressing fluorescent mScarlet protein at MOI 10:1 was either deposited on the top of the hNECs on Transwell membrane using (A) five 1 µl drops or (B) 200 μL of ALI medium, which was carefully removed without washing after 6 h of incubation. At indicated time points ZO-1 (yellow) was stained using an anti-ZO-1 antibody, followed by an anti-rabbit IgG-DYL405 conjugate, while cilia (cyan) were labeled with an anti-acetylated tubulin antibody, followed by an anti-mouse IgG-AF488 conjugate. Bacteria were visualized by mScarlet expression (magenta). Shown are maximum intensity projections (Z-max) from confocal Z-stack images. Images are representative of two independent experiments. Scale bars, 5 μm.

Article Snippet: The next day, the Transwell membranes were washed with PBST (3x 5 min) and incubated with the anti-rabbit IgG-DyLight-405 conjugate (Jackson Immunoresearch, Cat# 111-475-003) at a dilution of 1:300, and the anti-mouse donkey IgG-AF488 conjugate (Jackson Immunoresearch, Cat# 115-546-062) at a dilution of 1:500 in 1% BSA in PBST for 1 hour at RT.

Techniques: Expressing, Membrane, Incubation, Staining, Labeling, Bacteria