Isolation of chicken primordial germ cells by centrifugation in Ficoll gradient

Chicken primordial germ cells (PGCs) were isolated from embryo stage 27-28 HH. The cell suspension was obtained by digestion of whole chicken gonads in 0.25% trypsin/0.02% EDTA. A 0-26% Ficoll gradient was manually loaded into a centrifuge tube 15 ml type and a top was overlaid with 1 ml of the cell suspension. Centrifugation lasted 30 minutes at 170g. After that layers of 1 ml were drawn by the bottom puncturing of the tube. Each layer was analysed for the PGCs presence, their ratio was counted and a viability was determined by Trypan Blue. The most of the PGCs were found between 12%-14% layers and the ratio was 6.22%, viability decreased about 30%. After this procedure only 3.3% of all cells remained.