Intracellular cytokine detection based on flow cytometry in hemocytes from Galleria mellonella larvae
Anna Katarzyna Wrońska, Agata Kaczmarek, Mieczysława Irena Boguś
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. Therefore, a protocol for intracellular cytokine detection in Galleria mellonella larvae hemocytes based on flow cytometry has been developed. 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.
Steps
Sample preparation
Before bleeding, wash the larvae with distilled water (15 seconds) and then immerse briefly (5 seconds) in 70% (v/v) ethanol to sterilize their surfaces, thus reducing the chance of contamination of hemolymph samples.
Collect the hemolymph into sterile tube from the larvae through the incision made in the last proleg
100 μl of fresh hemolymph collected from ten larvae suspend in 100 μl of PBS+AB buffer
Centrifuge cells 300x g,4°C
Carefully remove the supernatant and resuspend the cell pellet in 100 μl of PBS+AB buffer. Repeat this step three times.
Fixation
Carefully remove the supernatant and resuspend the cell pellet in 500 μl of fixation buffer. Incubate for 0h 10m 0s Room temperature
Centrifuge at 300x g,4°C and remove the fixation buffer.
Wash fixed cells with PBS+AB buffer. Centrifuge at 300x g,4°C and discard the supernatant. Repeat this step three times.
Permeabilization
Resuspend the cell pellet in 500 μl of permeabilization buffer with 0.5% BSA (bovine serum albumin). Incubate for 0h 10m 0s Room temperature
Wash the fixed/permeabilized cells with permeabilization buffer. Centrifuge at 300x g,4°C and discard the supernatant. Repeat this step three times.
Staining
Dilute the primary antibody with permeabilization buffer to an optimal working concentration and resuspend the fixed/permeabilized cells with primary antibody solution. Incubate 4°C in the dark.
Centrifuge at 300x g,4°C and remove the supernatant.
Wash with permeabilization buffer and centrifuge at 300x g,4°C and discard the supernatant. Repeat this step three times.
Dilute the fluorescent-conjugated secondary antibody with permeabilization buffer for an optimal working concentration and resuspend the cell pellet with secondary antibody solution. Incubate for 1h 30m 0s Room temperature in the dark.
Wash with permeabilization buffer and centrifuge at 300x g,4°C and discard the supernatant. Repeat this step three times.
Resuspend cells in 1000 μl PBS+AB buffer. Before cytometric analysis, filter the resuspended cells using cell strainers, mesh size: 30μm.
