Separation of Penaeus vannamei haemocyte subpopulations by iodixanol density gradient centrifugation

Abstract

Methodologies for separation of immune cell subpopulations are essential tools in immunology studies. Up to date, only one methodology for separating crustacean haemocyte subpopulations using Percoll density gradient centrifugation has been described.In the present work, a new methodology to separate Penaeus vannamei haemocyte subpopulations was developed, using a two-step iodixanol density gradient centrifugation. P. vannamei haemolymph was collected with anticoagulant and centrifuged through a first gradient (densities from 1.063 to 1.109g/ml) for 10min at 2000g. Three bands were formed: two bands with lower density close together, and a third band with higher density. The first two were collected together whilst the third band was collected separately. The volume fraction in-between these bands contained dispersed cells and was also collected. The suspension containing the mixture of the first two bands was centrifuged through a second gradient (densities from 1.047 to1.087g/ml) for 15min at 2000g. Two bands were formed and collected individually. All the cell suspensions were used for in vitro culture (cell survival evaluation) and for evaluation of cell morphology by flow cytometry and light microscopy. Each of the three bands contained a major cell type with distinct morphology and behaviour. The dispersed cell fraction contained a mixture of two different cell types, which were distinct from the cell types in the bands. By order of appearance from the top of the gradient, the cell types were named: subpopulations (Sub) 1 (band 1), Sub 2 (band 2), Sub 3+4 (dispersed cells) and Sub 5 (band 3). The purity level (percentage of the major cell type) of Sub 1, 2 and 5 was 95.0±1.0%, 97.7±1.2% and 99.4±0.8%, respectively. Cells of Sub 2 showed the best survival time in vitro (up to 96h) followed by cells from Sub 1, Sub 3+4 and Sub 5. Phagocytic activity was detected in Sub 1 and 4.This methodology allowed the separation and characterization of five morphologically distinct and physiologically active P. vannamei haemocyte subpopulations, from which three were isolated with a very high degree of purity. Therefore, we consider this methodology a valuable alternative for the traditional crustacean haemocyte separation procedure in Percoll.