TITLE: Intracellular cytokine detection based on flow cytometry in hemocytes from Galleria mellonella larvae
AUTHORS: Anna Katarzyna Wrońska, Agata Kaczmarek, Mieczysława Irena Boguś

[DESCRIPTION]
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]
SECTION: Sample preparation
1. 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.

SECTION: Sample preparation
2. Collect the hemolymph into sterile tube from the larvae through the incision made in the last proleg


SECTION: Sample preparation
3. 100 μl of fresh hemolymph collected from ten larvae suspend in 100 μl of PBS+AB buffer
SECTION: Sample preparation
4. Centrifuge cells 300 x g, 5 min, 4 °C 

SECTION: Sample preparation
5. Carefully remove the supernatant and resuspend the cell pellet in 100 μl of PBS+AB buffer. Repeat this step three times.
SECTION: Fixation
6. Carefully remove the supernatant and resuspend the cell pellet in 500 μl of fixation buffer. Incubate for 10 min  Room temperature 

SECTION: Fixation
7. Centrifuge at  300 x g, 5 min, 4 °C  and remove the fixation buffer.
SECTION: Fixation
8. Wash fixed cells with PBS+AB buffer. Centrifuge at 300 x g, 5 min, 4 °C  and discard the supernatant. Repeat this step three times.

SECTION: Permeabilization
9. Resuspend the cell pellet in 500 μl of permeabilization buffer with 0.5% BSA (bovine serum albumin). Incubate for  10 min Room temperature 

SECTION: Permeabilization
10. Wash the fixed/permeabilized cells with permeabilization buffer. Centrifuge at 300 x g, 5 min, 4 °C  and discard the supernatant. Repeat this step three times.

SECTION: Staining
11. 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.

SECTION: Staining
12. Centrifuge at  300 x g, 5 min, 4 °C  and remove the supernatant.

SECTION: Staining
13. Wash with permeabilization buffer and centrifuge at 300 x g, 5 min, 4 °C and discard the supernatant. Repeat this step three times.

SECTION: Staining
14. 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 90 min Room temperature  in the dark.

SECTION: Staining
15. Wash with permeabilization buffer and centrifuge at 300 x g, 5 min, 4 °C  and discard the supernatant. Repeat this step three times.
SECTION: Staining
16. Resuspend cells in 1000 μl PBS+AB buffer. Before cytometric analysis, filter the resuspended cells using cell strainers, mesh size: 30μm.