Journal: F1000Research

Article Title: A co-culture model of the bovine alveolus

doi: 10.12688/f1000research.18696.2

Figure Lengend Snippet: List of reagents required for isolation of alveolar type II cells, generation of lentivirus and assembly of co-culture model.

Article Snippet: SV40 Lentivirus particles , AMS Biotechnology , LVP557-GP.

Techniques: Isolation, Plasmid Preparation, Expressing, Clone Assay, Transfection, Modification

Journal: F1000Research

Article Title: A co-culture model of the bovine alveolus

doi: 10.12688/f1000research.18696.2

Figure Lengend Snippet: For karyotype analysis, a metaphase spread was performed on Colcemid cell cycle arrested cultures for both BATII and B2AE, comparing with the wild-type ATII ( A ). The Bmi1 / hTERT transduced B2AE cell line was found to maintain the wild type bovine karyotype of 2n = 60, whilst the SV40 / hTERT transduced BATII cell line showed signs of genomic instability at both passages 14 and 23. This was reflected in a growth curve analysis of the two cell lines, which was performed by seeding primary ATII cells into a 96 well plate, comparing over 9 days with the BATII and B2AE cell lines ( B ), using the Cell Titre Aqueous One Assay (Promega). Later passages (p10 and p23) of BATII cells exhibited a considerably higher rate of proliferation than the wild type ATII, whilst the B2AE cell line at all passages was comparable with its wild type counterpart. Data shown as optical density read at 490 nm (OD 490nm ); Mean ± SD, n=3.

Article Snippet: SV40 Lentivirus particles , AMS Biotechnology , LVP557-GP.

Techniques:

Journal: F1000Research

Article Title: A co-culture model of the bovine alveolus

doi: 10.12688/f1000research.18696.2

Figure Lengend Snippet: Light microscopy images of the wild type ATII, SV40 / hTERT lentivirus transduced BATII and Bmi1 / hTERT lentivirus transduced B2AE were generated from cultures seeded into T75 cm 2 flasks and imaged on a Nikon Eclipse Ci upright microscope ( A ). Scale bar 50 μm. For IF, each cell type was seeded onto a Nunc Lab Tek II coverglass, before fixation and staining with the ATII marker SLC434A2 and vimentin, a marker of epithelial-mesenchymal transition ( B ). Both cell lines were more prone to colony formation than their wild-type counterpart, with BATII cells in particular forming 3D structures, whilst B2AE frequently formed ring-like colonies around a central ‘lumen’. Cells which exhibited a flattened morphology on the exterior of the colonies of both cell lines stained most strongly for vimentin, with one lone cell in the BATII image notable for the complete absence of SLC34A2 (middle panel, white arrow), which remained strong in the centre of the 3D colony. Scale bar = 100 μm.

Article Snippet: SV40 Lentivirus particles , AMS Biotechnology , LVP557-GP.

Techniques: Light Microscopy, Generated, Microscopy, Staining, Marker