Journal: Cytotechnology

Article Title: Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

doi: 10.1007/s10616-018-0272-y

Figure Lengend Snippet: Summary of genes used to immortalize BIEC-c4 cells and PCR conditions

Article Snippet: After 18 h incubation at 37 °C, cells in two wells were washed with sterile 1 × PBS and transfected with either the pSV3-neo (ATCC ® 37150) plasmid vector with SV40 gene or the pGRN145 plasmid vector with hTERT gene (ATCC ® MBA-141) using Lipofectamine ® 2000 reagent according to the manufacturer’s protocol.

Techniques:

Journal: Cytotechnology

Article Title: Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

doi: 10.1007/s10616-018-0272-y

Figure Lengend Snippet: Early passage bovine intestinal epithelial cells (BIEC-c4) and immortalized BIECs. a BIEC-c4 cells at passage 19. b SV40 immortalized BIECs at passage 36. c hTERT immortalized BIECs at passage 34. d HPV E6/E7 immortalized BIECs at passage 48. Pictures were taken at 20 × magnification and scale bar indicates 50 µm length

Article Snippet: After 18 h incubation at 37 °C, cells in two wells were washed with sterile 1 × PBS and transfected with either the pSV3-neo (ATCC ® 37150) plasmid vector with SV40 gene or the pGRN145 plasmid vector with hTERT gene (ATCC ® MBA-141) using Lipofectamine ® 2000 reagent according to the manufacturer’s protocol.

Techniques:

Journal: Cytotechnology

Article Title: Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

doi: 10.1007/s10616-018-0272-y

Figure Lengend Snippet: Immunocytochemical staining for cytokeratin and vimentin. IgG2a isotype control staining in BIEC-c4 cells at passage 32 ( a ). Anti-cytokeratin IgG2a Ab staining in BIEC-c4 cells at passage 51 ( c ), SV40-BIECs at passage 53 ( e ), hTERT-BIECs at passage 32 ( g ), and HPV-BIECs at passage 48 ( i ). IgM isotype control staining in BIEC-c4 cells at passage 53 ( b ). Anti-vimentin IgM Ab staining in BIEC-c4 cells at passage 53 ( d ), SV40-BIECs at passage 53 ( f ), hTERT-BIECs at passage 30 ( h ), and HPV-BIECs at passage 48 ( j ). Pictures were taken at 20 × magnification and scale bar indicates 50 µm length

Article Snippet: After 18 h incubation at 37 °C, cells in two wells were washed with sterile 1 × PBS and transfected with either the pSV3-neo (ATCC ® 37150) plasmid vector with SV40 gene or the pGRN145 plasmid vector with hTERT gene (ATCC ® MBA-141) using Lipofectamine ® 2000 reagent according to the manufacturer’s protocol.

Techniques: Staining

Journal: Cytotechnology

Article Title: Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

doi: 10.1007/s10616-018-0272-y

Figure Lengend Snippet: PCR products for SV40, hTERT, and HPV genes. PCR was performed following DNA extraction from each of the three immortalized BIEC cell types (hTERT-BIEC at passage 30, SV40-BIEC at passage 48, and HPV-BIEC at passage 72) using primers specific to each gene (Table ) and products were resolved on 1.5% agarose gel for the confirmation of hTERT gene in hTERT-BIEC DNA ( a ); SV40 gene in SV40-BIEC DNA ( b ); and E6/E7 gene in HPV-BIEC DNA ( c )

Article Snippet: After 18 h incubation at 37 °C, cells in two wells were washed with sterile 1 × PBS and transfected with either the pSV3-neo (ATCC ® 37150) plasmid vector with SV40 gene or the pGRN145 plasmid vector with hTERT gene (ATCC ® MBA-141) using Lipofectamine ® 2000 reagent according to the manufacturer’s protocol.

Techniques: DNA Extraction, Agarose Gel Electrophoresis

Journal: Cytotechnology

Article Title: Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

doi: 10.1007/s10616-018-0272-y

Figure Lengend Snippet: Immunocytochemical staining for SV40 and hTERT proteins. BIEC-c4 cells at passage 61 ( a ) and SV40-BIECs at passage 58 ( b ) were stained for SV40 protein with mouse SV40-specific monoclonal IgG2a Ab. BIEC-c4 cells at passage 34 ( c ) and hTERT-BIECs at passage 32 ( d ) were stained for hTERT protein with rabbit hTERT-specific polyclonal IgG Ab. Pictures were taken at 20 × magnification and scale bar indicates 50 µm length

Article Snippet: After 18 h incubation at 37 °C, cells in two wells were washed with sterile 1 × PBS and transfected with either the pSV3-neo (ATCC ® 37150) plasmid vector with SV40 gene or the pGRN145 plasmid vector with hTERT gene (ATCC ® MBA-141) using Lipofectamine ® 2000 reagent according to the manufacturer’s protocol.

Techniques: Staining

Journal: Cytotechnology

Article Title: Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

doi: 10.1007/s10616-018-0272-y

Figure Lengend Snippet: Growth kinetics analysis of BIEC-c4 cells and immortalized BIECs. a Growth curve for BIEC-c4 (passage # 51), SV40-BIEC (passage # 90), hTERT-BIEC (passage # 48), and HPV-BIEC (passage # 48). On day 0, 20,000 cells were plated on five 6-well plates for each cell type. Beginning on day 2, until day 6, cells were trypsinized every 24 h and counted using hemocytometer. b Analysis of doubling time for BIEC-c4 and immortalized BIECs. Mean doubling time was calculated from three independent experiments (n = 3) for each cell type. Two-tailed t test was performed to compare mean doubling time between BIEC-c4 and each of the three immortalized BIECs. P value < 0.05 was considered as statistically significant. Bars represent standard error of the mean

Article Snippet: After 18 h incubation at 37 °C, cells in two wells were washed with sterile 1 × PBS and transfected with either the pSV3-neo (ATCC ® 37150) plasmid vector with SV40 gene or the pGRN145 plasmid vector with hTERT gene (ATCC ® MBA-141) using Lipofectamine ® 2000 reagent according to the manufacturer’s protocol.

Techniques: Two Tailed Test

Journal: Cytotechnology

Article Title: Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

doi: 10.1007/s10616-018-0272-y

Figure Lengend Snippet: TLRs 1-10 expression in BIEC-c4 cells (passages 55–58) and immortalized BIECs. In this study, SV40-BIEC at passages 38–41, hTERT-BIEC at passages 32–36, and HPV-BIEC at passages 44–47 were used. Expression levels of each TLR was compared among the four BIEC cell types. Change in cycle threshold, ΔCt was used to calculate gene expression for each TLR. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta {\text{Ct}}$$\end{document} Δ Ct is calculated relative to HPRT-1 gene. P value < 0.05 was considered as statistically significant. Data presented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta {\text{Ct}}$$\end{document} Δ Ct from three independent experiments (n = 3) for each TLR. Bars represent standard error of the mean

Article Snippet: After 18 h incubation at 37 °C, cells in two wells were washed with sterile 1 × PBS and transfected with either the pSV3-neo (ATCC ® 37150) plasmid vector with SV40 gene or the pGRN145 plasmid vector with hTERT gene (ATCC ® MBA-141) using Lipofectamine ® 2000 reagent according to the manufacturer’s protocol.

Techniques: Expressing