2.2.1. Protocol for primary gut cells
1. Briefly immerse 10 day old pupae (see BEEBOOK paper on miscellaneous research methods (Human et al., 2013) for the method to obtain them) in 3 % H2O2 for surface sterilization.
2. Wash pupae with 1 X PBS (Table 1).
3. Cut off heads.
4. Fix thorax on a petri dish (35 mm in diameter) with wax, paraffin or silicon coated dish.
5. Cut proximal abdomen lateral and dorsal.
6. Open abdomen carefully.
7. Carefully add cold L15 medium (Table 1) supplemented with 10 % antibiotic/antimycotic solution (Sigma-Aldrich, A5955) to the opened abdomen.
8. Prepare a 24-well-plate with several wells filled with cold (4°C – 10°C) L15 medium.
9. Extract gut and place it in a medium-filled well of the 24-well plate (see step 8).
10. Prepare several guts following the above outlined procedure and place up to 10 guts into one well.
11. Remove medium carefully.
12. Add 1 ml of enzyme solution (L15 medium (Table 1), 0.05 % trypsin (Invitrogen, 15400054) and 0.5 % collagenase/dispase (Roche, 10269638001)) to disintegrate the tissue.
13. Incubate plate with gentle shaking at 4°C for 1 hour.
14. Incubate plate with gentle shaking at 30°C for 1 hour.
15. Incubate plate with gentle shaking at 4°C for 1 hour.
16. Transfer gut/cell-suspension to a 1.5 ml-reaction tube.
17. Centrifuge for 3 min with 300 rcf (Eppendorf 5415 R).
18. Remove supernatant and gently resuspend the pellet in L15 medium (40 µl per gut) to dissociate the cells.
19. Dispense 40 µl of cell suspension per well in a 96-well plate or per well of a chamber slide (8 well glass slide, VWR).
20. Incubate 20 min at 33°C to allow cell attachment.
21. Add 60 µl pre-warmed (37°C) BM3 medium (Table 1) supplemented with 10 % antibiotic/antimycotic solution per well.
22. Incubate in an incubator suitable for insect cell culture [cooling incubator] for 24 h at 33°C.
23. Discard medium
24. Add 100 µl fresh, pre warmed BM3 medium.
Cells remain vital
for several weeks or even months.