(185e) Separation of Hemocrit from Hemolymph Collected from Individual Drosophila

Although Drosophila melanogaster is an important animal model for many human diseases, the small size of Drosophila typically limits the volume of sample that can be collected which complicates obtaining chemical content data. Work in our lab has focused on the collection and chemical analysis of the tens of nanoliter volumes of hemolymph that can be collected from individual Drosophila. The blood-like hemolymph plays roles of chemical delivery and waste removal and also contains hemocyte cells that provide an immune response. All previous work has employed whole-hemolymph, plasma and hemocyte content, for chemical analysis including quantitation of amino acids and proteomics. The goal in this work is to separate the cellular content from the hemolymph plasma for subsequent analysis. Following collection of hemolymph from individual Drosophila, hemolymph is diluted to slow the clotting reaction. A number of strategies were tested for separation of hemocrit including filtering schemes and centrifugation. Hemocyte content was greatly reduced by centrifugation by not by filtering as determined by microscopic fluid analysis following cellular staining. Microscopy of hemocrit containing sample revealed cell types as identified by staining, shape and abundances in accordance with previously reported literature. Amino acid content of hemolymph plasma was compared to whole hemolymph via an electrophoretic assay. Following reaction of hemolymph primary amine components with 3-(4-carboxybenzoyl)quinoline-2-arboxaldehyde electrophoretic separation revealed lower plasma levels compared to whole hemolymph. Amino acid content analysis of hemocrit fraction will also be discussed. Separation of hemocrit from hemolymph demonstrates an ability to further isolate specific Drosophila tissues. The ability to isolate plasma may simplify assay development for hemolymph-mediated chemical signaling whereas hemocyte collection may allow new immunologic experimentation.