Izvorni znanstveni članak

https://doi.org/10.24099/vet.arhiv.0956

Optimization of the method for isolation of epithelial cells from the non-glandular part of the rat stomach for flow cytometry

Gordana Joksić ; „VINČA” Institute of Nuclear Sciences - National Institute of thе Republic of Serbia, University of Belgrade, Begrade, Republic of Serbia
Jelena Filipović Tričković ; „VINČA” Institute of Nuclear Sciences - National Institute of thе Republic of Serbia, University of Belgrade, Begrade, Republic of Serbia
Mileva Mićić ; Institute for Medical Research, University of Belgrade, Belgrade, Republic of Serbia
Ivana Joksić ; „VINČA” Institute of Nuclear Sciences - National Institute of thе Republic of Serbia, University of Belgrade, Begrade, Republic of Serbia; Clinic for Gynecology and Obstetrics Narodni Front, Belgrade, Republic of Serbia
Ana Valenta Šobot ; „VINČA” Institute of Nuclear Sciences - National Institute of thе Republic of Serbia, University of Belgrade, Begrade, Republic of Serbia
Miroslav Demajo ; „VINČA” Institute of Nuclear Sciences - National Institute of thе Republic of Serbia, University of Belgrade, Begrade, Republic of Serbia

Sažetak

Traditional methods in cell proliferation studies are based on immunohistochemical detection of proliferating cells in the target tissue. Since they are time consuming, optimization of novel, more efficient methods is important for large scale proliferation studies. In this study, we aimed to optimize the isolation of single epithelial rat forestomach cells for flow cytometry. As a marker of cellular proliferation we used the Ki-67 antibody to detect this nuclear protein expressed in proliferating cells. We also performed immunohistochemical detection of Ki-67 positive cells and propidium iodide staining to validate the results. 3-tert- butyl -4-hydroxyanisole was used as the positive control to ensure cellular proliferation. The results showed that isolation of epithelial cells with collagenase, trypsin and cell strainer ensures great cell viability (>95%) and the purity of the samples. Flow cytometry and immunostaining with the Ki-67 antibody indicated that 3-tert- butyl-4-hydroxyanisole treatment leads to a significant increase in proliferation. A significant positive correlation was observed between the results obtained by immunohistochemistry and flow cytometry, but the flow cytometric data had a smaller measurement error, suggesting the equal sensitivity and greater accuracy of this method. Propidium iodide staining showed that the percentage of cells in the G2+S phase of the cell cycle correlated positively with the percentage of Ki-67 positive cells assessed by flow cytometry, indicating that Ki-67 positive cells reflect an active dividing cell pool. We conclude that the isolation of forestomach epithelial cells described is a simple and reliable method for obtaining viable cells for use in flow cytometry. Compared to immunohistochemistry, flow cytometric detection of the Ki-67 antigen is equally sensitive, but much faster and provides more accurate results.

Ključne riječi

Ki-67; epithelial cell isolation; flow cytometry; non-glandular part of the rat stomach; cell proliferation

Hrčak ID:

245824

URI

https://hrcak.srce.hr/245824

Datum izdavanja:

10.11.2020.

Podaci na drugim jezicima: hrvatski

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