TY - JOUR
T1 - Use of flow cytometry to quantify mouse gastric epithelial cell populations
AU - Zavros, Yana
AU - Van Antwerp, Mary
AU - Merchant, Juanita L.
N1 - Funding Information:
Y.Z. is a Howard Hughes research associate. J.L.M. is an investigator of the Howard Hughes Medical Institute. This work was supported in part by Public Health Service grant DK-45729 to J.L.M. Flow cytometry was performed with support from the University of Michigan Comprehensive Cancer Center (P30 CA 46592).
PY - 2000
Y1 - 2000
N2 - Flow cytometry provides the opportunity to quantify cell populations within a total cell suspension. The quality of flow cytometry is strongly dependent on the isolation of intact viable cells. However, techniques to isolate mouse gastric cells for flow cytometry have not been evaluated. The objective of this study was to develop an effective method for isolating intact viable cells from mouse gastric tissue for flow cytometry. Cells were isolated from mouse stomach and spleen by either enzymatic separation or mechanical dissociation. A Percoll density gradient was used to separate viable cells from cellular debris. Cells were labeled with fluorescently tagged ligand or antibody and analyzed by flow cytometry. According to propidium iodide staining, there was a higher percentage of viable cells after mechanical dissociation (10-20%) compared to enzymatic separation (1%). After Percoll centrifugation there was a further increase in the percent of viable cells (50-80%). Gastrin (G), somatostatin (D), and parietal cells represented 0.6%, 3%, and 8% of the total epithelial cell population, respectively. T and B lymphocytes made up 4% and 2% in the gastric mucosa. Dissociated splenocytes were comprised of 20% T cells and 14% B cells. The ability to reliably resolve a cellular fraction that comprises only 0.6% of the input marks a substantial improvement over morphometric methods. Therefore, mechanical dissociation of the stomach followed by use of a Percoll gradient is the preferred method for isolating viable intact gastric epithelial cells for flow cytometry.
AB - Flow cytometry provides the opportunity to quantify cell populations within a total cell suspension. The quality of flow cytometry is strongly dependent on the isolation of intact viable cells. However, techniques to isolate mouse gastric cells for flow cytometry have not been evaluated. The objective of this study was to develop an effective method for isolating intact viable cells from mouse gastric tissue for flow cytometry. Cells were isolated from mouse stomach and spleen by either enzymatic separation or mechanical dissociation. A Percoll density gradient was used to separate viable cells from cellular debris. Cells were labeled with fluorescently tagged ligand or antibody and analyzed by flow cytometry. According to propidium iodide staining, there was a higher percentage of viable cells after mechanical dissociation (10-20%) compared to enzymatic separation (1%). After Percoll centrifugation there was a further increase in the percent of viable cells (50-80%). Gastrin (G), somatostatin (D), and parietal cells represented 0.6%, 3%, and 8% of the total epithelial cell population, respectively. T and B lymphocytes made up 4% and 2% in the gastric mucosa. Dissociated splenocytes were comprised of 20% T cells and 14% B cells. The ability to reliably resolve a cellular fraction that comprises only 0.6% of the input marks a substantial improvement over morphometric methods. Therefore, mechanical dissociation of the stomach followed by use of a Percoll gradient is the preferred method for isolating viable intact gastric epithelial cells for flow cytometry.
KW - Gastric epithelial cells
KW - Gastrin
KW - Helicobacter pylori
KW - Parietal cells
KW - Sornatostatin
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U2 - 10.1023/A:1005514422187
DO - 10.1023/A:1005514422187
M3 - Article
C2 - 10877237
AN - SCOPUS:0034089103
SN - 0163-2116
VL - 45
SP - 1192
EP - 1199
JO - Digestive diseases and sciences
JF - Digestive diseases and sciences
IS - 6
ER -