primary human lung microvascular endothelial cells lmec  (PromoCell)

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: To evaluate replication efficiency, lung microvascular endothelial cells (LMECs) were plated on the (A) apical or (B) basolateral side of a transwell filter. LMECs were inoculated with pH1N1, H1N1, or H3N2 virus at MOI 1 and at the indicated time points supernatants of the apical as well as basolateral side were harvested and virus titers were determined by endpoint titration. Infection efficiency was determined by immunofluorescence staining. LMECs plated on the (C) apical and (D) basolateral were inoculated with pH1N1, H1N1, or H3N2 virus. Cells were fixed 24 hours post inoculation and stained for the endothelial cell marker Vascular Endothelial-Cadherin (VE-CAD, magenta) and influenza A virus nucleoprotein (NP (green). Hoechst (blue) was used to visualize nuclei. (E) Percentage of infection determined by flow cytometry at 24 and 72 hours post inoculation. (F) Viral RNA genome copies were quantified by quantitative real time PCR at indicated time points. Data represent mean +/- standard deviation (SD) from at least three independent experiments performed in biological duplicates and flow cytometry was performed in biological triplicates. A one-way ANOVA multiple comparison test was used to compare groups (*< 0.05, **<0.01, ***<0.005). Scale bar: 20 μm.

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Virus, Titration, Infection, Immunofluorescence, Staining, Marker, Flow Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: (A) Well-differentiated airway organoids at air-liquid interface (AO at ALI) in co-culture with lung microvascular endothelial cells (LMECs) were inoculated with pH1N1, H1N1 or H3N2 virus at MOI 1. At the indicated timepoints virus titers were determined in the supernatants of the apical and basolateral compartments. (B) Detection of influenza A virus (IAV) nucleoprotein (NP) by immunohistochemistry of the AO at ALI-LMEC co-cultures 24 hours post inoculation (C) Hematoxylin and eosin (H&E) staining of the co-cultures 72 hours post inoculation (scale bar 20 μm) (D) At 72 hours post inoculation well-differentiated AO at ALI were stained for IAV NP (green), the cilia marker acetylated-α-tubulin (cyan) and the tight-junction marker Zona occludin 1 (ZO-1, magenta) on the apical compartment of the transwell. The basolateral compartment containing the LMECs was stained for IAV NP (green) and the endothelial cell marker Vascular-Endothelial Cadherin (VE-CAD, magenta). In both cases the nuclei were visualized with Hoechst (scale bar 20 μm). (E) Epithelial cells (AO at ALI) or endothelial-epithelial co-cultures were inoculated with pH1N1, H1N1 or H3N2 virus at MOI 1. At 24 hours post-inoculation cytokines were measured in the apical compartment using the Legendplex assay. Data represented here show individual data points of cytokines derived from three independent experiments performed in biological duplicates and the mean +/- standard deviation (SD) is depicted. Mock of each condition was subtracted from the values of virus infected cells. Statistical significance was determined with Students-T-test (*<0.05, **<0.01, ***<0.005, ****<0.001).

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Co-Culture Assay, Virus, Immunohistochemistry, Staining, Marker, Derivative Assay, Standard Deviation, Infection

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: Lung microvascular endothelial cells (LMECs) or differentiated airway organoids at air-liquid interface in co-culture with LMECs were inoculated with pH1N1 virus at MOI 1. (A) percentage of infection and (B) viral genome copies in LMEC single cultures compared to co-cultures were determined by flow cytometry or qRT-PCR at 24 hours post-inoculation. Data represented here show pooled data of virus titers derived from three independent experiments performed in biological duplicates and the mean +/- standard deviation is depicted. A student T-test was used to compare groups (*<0.05, **<0.01, ***<0.005, ****<0.001).

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Co-Culture Assay, Virus, Infection, Flow Cytometry, Quantitative RT-PCR, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: (A) Endothelial cells (lung microvascular endothelial cells), epithelial cells (airway organoids at air-liquid interface) and endothelial-epithelial co-cultures were inoculated with pH1N1, H1N1 and H3N2 virus at MOI 1. At 24 hours post-inoculation cytokines were measured in the basolateral compartment using a Legendplex assay. Data represented here show individual data points of cytokines derived from three independent experiments performed in biological duplicates and the mean +/- standard deviation is depicted. Mock of each condition was subtracted from the values of virus inoculated cultures. Statistical significance was determined with One-Way Anova and each group was compared to each other (*<0.05, **<0.01, ***<0.005, ****<0.001).

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Virus, Derivative Assay, Standard Deviation