Dissociation of chicken blastoderm for examination of apoptosis and necrosis by flow cytometry

Abstract

We describe a method of isolating blastodermal cells for characterization through flow cytometry. Particular attention was placed on cell viability and integrity issues faced by conventional protocols. The method allowed us to examine mechanisms behind cellular death. Our protocol was optimized by the spatial resolution of the ImageStream multispectral imaging flow cytometer. Overall, the technique provides both quantitative and qualitative information on blastodermal cells. The methodology was applied to the current biological problem in which prolonged (14 d) versus short-term (4 d) layer egg storage reduces embryo viability. Data were obtained between 3 egg storage treatments (unstored, 4 d, and 14 d); the data were analyzed by the PROC MIXED model procedure of SAS at P < or = 0.05 and least squares means separated by the PDIFF procedure of SAS. The results showed that egg storage increases the rate of cell death by both apoptosis and necrosis. Importantly, our study showed higher percentages of necrosis and late apoptosis in long-term versus short-term stored eggs. The percentage of live cells decreased significantly when eggs were stored for 14 d (71.42 + or - 3.36%) compared with eggs stored for 4 d (83.58 + or - 2.15%). The percentage of early apoptotic cells was not significantly different between the 2 treatments. The percentage of necrotic cells and late apoptotic-necrotic cells was higher in eggs stored for 14 d (16.75 + or - 1.73%; 7.36 + or - 1.53%) versus eggs stored for 4 d (3.56 + or - 1.64%; 2.31 + or - 1.52%), respectively. This could negatively affect embryo survival because of the potential effect that necrosis has on surrounding tissue integrity. The technique will be particularly relevant in studies requiring single cells from chicken blastoderms or as a basis to characterize genes that regulate apoptosis in avian species.