FLUORESCEIN LABELLED ANNEXIN V STAINING LIMITATIONS FOR DETERMINATION OF BLASTODERMAL CELLS APOPTOSIS

Abstract

It is known that blastoderm contains not only blastodermal cells (BCs) but also cellular “debris”. These particles include yolk granules and lipid droplets. Some authors used AnnexinV (AnV) staining technique for determination of cell apoptosis. However, AnV is a protein with high affinity to lipids. So the aim of our study was to demonstrate the AnV binding in the blastoderm and yolk cell suspensions. Cell populations were obtained from fertilized chicken eggs. The blastoderms were isolated using filter paper ring method and mechanically fragmented. After that, samples were stained with AnV-FITC and analyzed by fluorescent microscopy (FM) and flow cytometry (FC). FM showed non-specific binding of AnV-FITC on the yolk and lipid particles. Therefore, the FC method using specific nuclear dye DRAQ5 was used to separate BCs from cellular debris. Only 1.82 ± 0.27 % cells were DRAQ5 positive. On the other hand, increase of total events (21 ± 3.30 %) of AnV positive cells without DRAQ5 dye was recorded. Moreover, the affinity of yolk to AnV by FC was also observed (10.45 ± 2.59 %). Significant differences among the groups (P < 0.001) were recorded. This trend was caused by non-specific binding of AnV to the lipid and yolk particles. So it is necessary to stain suspension with nuclear dye to separate BCs than co-stained with some marker of apoptosis. Based on the results we can summarized that AnV staining is inappropriate technique for apoptosis of BCs, however, using nuclear dye we can prevent of non-specific binding.