A method to isolate parasitized macrophages from spleen of Leishmania donovani infected mice

In order to characterize the process of systemic infection of C57BL/6 mice with the obligate intracellular parasitic protozoon Leishmania donovani in the spleen the available methods of isolation of splenic macrophages had to be substantially improved. This was necessary because heavily prasitized macrophages are extremely sensitive to mechanical disruption in the course of various standard homogenization procedures. Fully intact macrophages were also prerequisit for further functional testing in comparison with macrophages from bealthy animals. The excised spleen was carefully peeled and then exposed to pronase (Boehringer) in order to loosen cell-to-cell contact (0.15 w/v pronase in Hank's solution, 30 min. 37 degree C under continuous stirring). Leishmania-parasitized macrophages were then isolated in a percoll gradient (30-60 % in steps, 1700 rpm, 30 min at room temperature). All isolation steps took place in the presence of pancreatic DNAse I (100 myg/ml). The yield of macrophages amounted to 30-40% of all nucleated splenic cells. The described method of isolation of parasitzized splenic macrophages is distinguished by the very gentle handling of these vulnerable cells. Viable macrophages with more than 40 amastigote Leishmania donovani organisms/cell have been isolated from the spleen in large amounts (21 d p.i.). These cells could then be subjected to immunological testing in vitro. It could be shown for instance, that parasitized macrophages from C57BL/6 mice isolated at the peak of infection (28 d p.i.) expressed enhanced spontaneous activity for promastigote Leishmania donovani targets