Separation of young and mature thrombocytes by a novel immuno-selection method

Abstract

Our earlier studies on the structural and functional properties of zebrafish thrombocytes have shown that they have many similarities to mammalian platelets. We have also shown that zebrafish have both young and mature thrombocytes as do mammalian platelets. In addition, we have distinguished young thrombocytes from mature thrombocytes microscopically using lipophilic DiI-C18, and have shown that young thrombocytes have higher GPIIb receptor levels. However, at present, there is no immunoselection method to separate young thrombocytes from mature thrombocytes in order to study differences among them, such as mRNA expression levels of thrombocyte specific genes. We developed a novel technique employing specific biotinylated anti-Cy3 antibody against the chromophore of DiI-C18 and using streptavidin magnetic beads to separate young thrombocytes from mature thrombocytes. Our technique separates and differentiates young and mature thrombocytes from whole blood. This method is specific and is effective with small amounts of blood.

Fig. 1

Comparison of the structures of DiI-C18 (top) and Cy3 (bottom) dyes. Note the similarity between the heterocyclic chromophores of these two dyes.

Fig. 2

Principle of thrombocyte isolation using magnetic beads. Y, young thrombocyte; M, mature thrombocyte; R, red cell; N, neutrophil; B, biotin labeled antibody; S, streptavidin beads attached to magnet. IgG/Cy3 and IgG/GPIIb are represented as Y shaped structures. Thrombocyte and neutrophil are shown in shaded circles. The triangular and square structures on the shaded circles represent Cy3 and GPIIb respectively. Red cells are shown in shaded ovals.

Fig. 3

Immunofluorescence images of blood cells. A, images showing blood cells incubated with anti-Cy3 antibody kept under a cover slip. Left panel, bright field image; middle panel, fluorescence image of the DiI labeled thrombocyte; right panel, fluorescence image of the streptavidin FITC labeled thrombocyte. B, images showing blood smears probed with biotinylated anti-GPIIb antibody followed by streptavidin FITC. Left panel, bright field image; right panel, fluorescence image of the streptavidin FITC labeled thrombocyte. Arrows indicate the thrombocytes.

Fig. 4

Slide showing streptavidin magnetic beads after pulling the DiI labeled young thrombocytes. A, bright field images and B, fluorescence images.

Fig. 5

Purity of the isolated young thrombocytes. (A) Whole blood RNA control showing positive amplification for EF1-α (labeled as EF-α), Pu.1, GPIIb, and β-globin (labeled as β glo) genes. (B) Young thrombocyte RNA showing positive amplification for EF1-α and GPIIb genes.

Fig. 6

Presence of GPIIb transcripts in mature thrombocytes. Left and right panels show the positive amplification for EF1-α and GPIIb in both young and mature thrombocytes, respectively.