Intracellular cytokine detection based on flow cytometry in hemocytes from Galleria mellonella larvae: A new protocol

Abstract

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.

Characterising phagocytes and measuring phagocytosis from liveGalleria mellonellalarvae

“…In contrast with previous reports demonstrating separable sub-populations of fixed Galleria hemocytes based on relative size and internal complexity by flow cytometry [28], we found that live hemocytes, subjected to a minimal processing method, behaved as a single population. Which instead corroborates more recent methods to analyse fixed hemocytes by flow cytometry [27]. Even with the use of 4% PFA (Figure 2A) we failed to observe distinct hemocyte groups based on FSC and SSC.…”

“…This is supported by the observation that one of the assigned cell gates from that study appears in the same vicinity as our described debris gate - which we have conclusively demonstrated does not contain viable hemocytes (Figure 2 A and B). Further to this, others also report that the use of fixatives, as well as altering centrifugation speed, had an overall effect on cell morphology – as shown by clear differences in the FSC vs SSC scatter plots [27]. However, we do not rule out the possibility that the hemocyte landscape changes developmentally, as the larvae progress towards pupation, possibly resulting in a more complex distribution of cells the onset of metamorphosis [23].…”

“…to Galleria, but flow plots vary widely across publications and methods use generally fixed, rather than live, immune cells [26][27][28][29]. Though fixation is vital for the detection of intercellular antigens via antibody staining, and for the attenuation of hazardous biological materials, fixatives have been shown to affect both relative cell size and granularity when analysed by flow cytometry [30].…”

Galleria mellonella–intracellular bacteria pathogen infection models: the ins and outs

“…However, progress is being made, e.g. in the recent description of intracellular interferon-gamma detection in larval haemocytes (Wrońska et al 2022 ). Detection was based on the cross-reactivity of rabbit monoclonals recognizing human interferon gamma with G. mellonella interferon gamma.…”

The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae

“…Flow cytometry analysis was performed according to a protocol previously developed by our team for the determination of cytokine-like proteins in insect hemocytes ( 28 ). The hemolymph taken from both untreated and fungus-treated larvae was centrifuged (400xg, 10 min) and washed with PBS.…”