Dissociation of insect Malpighian tubules into single, viable cells.

Abstract

The Malpighian tubules of insects are generally composed of more than one cell type. In the hemipteran Rhodnius prolixus, the tubules are divided into two regions, termed the upper and lower tubules, each of which is composed of a distinct cell type. In the dipteran Aedes taeniorhynchus, primary and stellate cells are interspersed throughout the length of the tubules. We report here techniques for the dissociation of the Malpighian tubules of both of these species into single cells. Tubules are removed from the insect and placed for 1 h in insect Ringer containing elastase (Sigma, type III) at 4 mg/ml. This treatment fully removes the basal lamella. Mild agitation by hand produces a suspension of single cells, which remain viable as determined by Trypan Blue exclusion. Isolated cells have been maintained in cell culture for one week. Using light and scanning electron microscopy, upper and lower tubule cells of Rhodnius and primary and stellate cells of Aedes can be distinguished on the basis of size, shape, microvillar length, and the presence or absence of intracellular crystals.