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Molecular Characterization of Gastric Epithelial Cells Using Flow Cytometry.

International journal of molecular sciences (2018-04-13)
Kevin A Bockerstett, Chun Fung Wong, Sherri Koehm, Eric L Ford, Richard J DiPaolo
ABSTRACT

The ability to analyze individual epithelial cells in the gastric mucosa would provide important insight into gastric disease, including chronic gastritis and progression to gastric cancer. However, the successful isolation of viable gastric epithelial cells (parietal cells, neck cells, chief cells, and foveolar cells) from gastric glands has been limited due to difficulties in tissue processing. Furthermore, analysis and interpretation of gastric epithelial cell flow cytometry data has been difficult due to the varying sizes and light scatter properties of the different epithelial cells, high levels of autofluorescence, and poor cell viability. These studies were designed to develop a reliable method for isolating viable single cells from the corpus of stomachs and to optimize analyses examining epithelial cells from healthy and diseased stomach tissue by flow cytometry. We performed a two stage enzymatic digestion in which collagenase released individual gastric glands from the stromal tissue of the corpus, followed by a Dispase II digestion that dispersed these glands into greater than 1 × 10⁶ viable single cells per gastric corpus. Single cell suspensions were comprised of all major cell lineages found in the normal gastric glands. A method describing light scatter, size exclusion, doublet discrimination, viability staining, and fluorescently-conjugated antibodies and lectins was used to analyze individual epithelial cells and immune cells. This technique was capable of identifying parietal cells and revealed that gastric epithelial cells in the chronically inflamed mucosa significantly upregulated major histocompatibility complexes (MHC) I and II but not CD80 or CD86, which are costimulatory molecules involved in T cell activation. These studies describe a method for isolating viable single cells and a detailed description of flow cytometric analysis of cells from healthy and diseased stomachs. These studies begin to identify effects of chronic inflammation on individual gastric epithelial cells, a critical consideration for the study of gastric cancer.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dispase® II, protease
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Collagenase from Clostridium histolyticum, Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use
Sigma-Aldrich
Gentamicin sulfate, meets USP testing specifications, powder
DTT, Cytiva 17-1318-01, pack of 1 g