Invertebrates are becoming increasingly popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. However, the use of insects as research models requires the development of methods for working with hemocytes. The aim of this study was to develop a protocol for intracellular cytokine detection in Galleria mellonella larvae hemocytes based on flow cytometry. It describes the anticoagulant composition of the buffer, the optimal conditions for hemocyte permeabilization and fixation, as well as the conditions of cell centrifugation to prevent cell disintegration. A key element is the selection of staining conditions, especially the length of the incubation time with the primary antibody, which turned out to be much longer than recommended for mammalian cells. The development of these individual steps allowed for the creation of a reproducible protocol for cytokine detection using flow cytometry in wax moth hemocytes. This will certainly facilitate the development of further protocols allowing for wider use of insect cells in immunological research.

A range of analytical methods (GC-MS, LC-MS, voltammetry, microbiological and microscopic techniques, PCR) was used to assay a range of potential chemical and biological contaminants in soil and dandelion samples. The results provide the first comprehensive safety analysis of dandelion as a herbal product. Samples were collected from three different sites in Poland where the local population collects dandelion plants for their own consumption: Rudenka (a mountain meadow in the European Ecological Network of Natura 2000 protection area, free of agrotechnical treatments for over 30 years), Warszawa 1 (dense single-family housing with heavy traffic), and Warszawa 2 (recreation area with heavy traffic near a coal-fired heat and power plant). The assays of heavy metals and other chemical pollutants (PAHs, PCBs, dioxins, pesticides, mycotoxins) confirm that all collected soil and dandelion samples were chemically pure; however, 95 species of pathogenic bacteria were detected, including “carnivorous” Vibrio vulnificus, zoonotic Pasteurella pneumotropica, Pasteurella canis, Staphylococcus pseudintermedius, Staphylococcus lentus and Francisella tularensis as well as 14 species of pathogenic fungi and one protozoan parasite (Giardia intestinalis). The discovery of septicemia agents V. vulnificus, Fusobacterium mortiferum and Rahnella aquatilis in the soil surrounding dandelion roots and in the flowers, G. intestinalis in dandelion leaves and roots samples, all collected in Warsaw, is highly disturbing. This finding underlines the need for increased caution when collecting dandelion in densely populated areas with a large population of pets. Thorough washing of the harvested plants is necessary before using them for consumption, especially in the case of making salads from fresh dandelion leaves, which is becoming increasingly popular among people leading healthy and an environmentally friendly lifestyle.