Use of a two-step Percoll (R) gradient for separation of loggerhead sea turtle peripheral blood mononuclear cells

In order to determine a suitable procedure for isolating peripheral blood m ononuclear cells PBMCs) from loggerhead sea turtles (Caretta caretta, blood was collected using three different anticoagulants (sodium heparin, sodium citrate or potassium EDTA) and separated using a single step commercially- prepared arabinogalactan gradient of 1.077 g/ ml density or multiple step P ercoll gradients between 1.053 and. 1.076 g/ml density (40-60% stock isoton ic Percoll suspension). Heparinized blood centrifuged over a two-step 45/55 % (1.059/1.070 g/ml) Percoll gradient yielded 99 to 100% mononuclear cells at the 45/55% interface. Mononuclear cell viability ranged from 85 to 97% w ith cell yields up to 9:2 x 10(6) cells/ mL. An unexpected finding was a po pulation of low density granulocytes migrating to 40% (1.053 g/ml) and 35% Percoll layers in the multiple step gradients. These granulocytes could be eliminated from the PBMC preparation by use of the two-step 45/55% Percoll gradient. Isolated PBMCs can be used for cellular immunology and toxicology studies on these threatened marine organisms for which other tissues can u sually be obtained only sporadically from post-mortem specimens.