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1 July 2000 Use of a Two-step Percoll® Gradient for Separation of Loggerhead Sea Turtle Peripheral Blood Mononuclear Cells
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Abstract

In order to determine a suitable procedure for isolating peripheral blood mononuclear 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 Percoll gradients between 1.053 and 1.076 g/ml density (40–60% stock isotonic 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% with cell yields up to 9.2 × 106 cells/mL. An unexpected finding was a population of low density granulocytes migrating to 40% (1.053 g/ml) and 45% 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 usually be obtained only sporadically from post-mortem specimens.

Harms, Keller, and Kennedy-Stoskopf: Use of a Two-step Percoll® Gradient for Separation of Loggerhead Sea Turtle Peripheral Blood Mononuclear Cells
Craig A. Harms, Jennifer M. Keller, and Suzanne Kennedy-Stoskopf "Use of a Two-step Percoll® Gradient for Separation of Loggerhead Sea Turtle Peripheral Blood Mononuclear Cells," Journal of Wildlife Diseases 36(3), 535-540, (1 July 2000). https://doi.org/10.7589/0090-3558-36.3.535
Received: 13 May 1999; Published: 1 July 2000
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