Protocol detail

Isolation and Characterization of Intestinal Epithelial Cells

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The IECs were isolated from jejunum (mid gut) using a modified protocol adapted from Paim et al. [25 (link),34 (link),45 (link)]. The viability and numbers of IECs were determined by the trypan blue exclusion method (70–80%). qRT-PCR was performed using equal amounts of total RNA (75 ng) with Power SYBR Green RNA-to-CT 1 step RT-PCR kit (Applied Biosystems, Foster, CA, USA). The gene-specific primers for enteroendocrine cells (chromogramin A (CgA)), goblet cells (mucin 2 (MUC2)), transient amplifying progenitor cells (proliferating cell nuclear antigen (PCNA)), intestinal epithelial stem cells (transcription factor SRY-box9 (SOX9)), enterocytes (villin), and β-actin were based on previously published data [25 (link),46 (link),47 (link),48 (link)]. Relative gene expressions of CgA, MUC2, PCNA, SOX9, and villin were normalized to β-actin and expressed as fold change using the 2^−ΔΔCt method [49 (link)].

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Michael H., Miyazaki A., Langel S.N., Amimo J.O., Kick M.K., Chepngeno J., Paim F.C., Fischer D.D., Rajashekara G., Saif L.J, & Vlasova A.N. (2022). Escherichia coli Nissle 1917 Enhances Efficacy of Oral Attenuated Human Rotavirus Vaccine in a Gnotobiotic Piglet Model. Vaccines, 10(1), 83.

Publication 2022

Power SYBR Green RNA-to-CT 1 step RT-PCR kit

Actin Enterocytes Enteroendocrine cells Epithelial cells Gene Gene expressions Goblet cells Intestinal Jejunum Mucin 2 Primers Progenitor cells Proliferating cell nuclear antigen Rt pcr Stem Sybr green Transcription factor Transient Trypan blue Villin

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Variable analysis

independent variables

Modified protocol adapted from Paim et al. [25, 34, 45] for isolating IECs from jejunum (mid gut)

dependent variables

Viability and numbers of IECs determined by the trypan blue exclusion method (70–80%)
Relative gene expressions of CgA, MUC2, PCNA, SOX9, and villin normalized to β-actin and expressed as fold change using the 2^−ΔΔCt method

control variables

Equal amounts of total RNA (75 ng) used for qRT-PCR
Gene-specific primers for enteroendocrine cells (CgA), goblet cells (MUC2), transient amplifying progenitor cells (PCNA), intestinal epithelial stem cells (SOX9), enterocytes (villin), and β-actin based on previously published data [25, 46, 47, 48]

controls

No positive or negative controls were explicitly mentioned.

Annotations

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