2.1.2. Protocol for adult cells
For isolation of neurons from adult animals (age 1-3 days, see BEEBOOK paper on miscellaneous research methods (Human et al., 2013) for the method to obtain them) the procedure follows a slightly modified protocol.
1. Collect the brain parts of interest, e.g., mushroom bodies, antennal or optical lobes (it is recommended to take at least 5 animals to obtain enough cells).
2. Incubate in accutase (PAA, #L11-007) for 30 min at RT.
Using collagenase/dispase (Roche, 10269638001) (1mg/ml calcium-free ringer solution (Table 1)) for 30 min is also possible. However, this requires pre-tests to determine the temperature for optimal results (≈30-36°C).
3. Carefully resuspend the brain tissue 5 times with a pipette (1 ml pipette tip).
4. Incubate about 15-20 sec to allow for sedimentation of the neuropil parts.
5. Transfer the supernatant with the neurons into a 1.5 ml reaction tube
6. Centrifuge at 1,100 rcf for 3 min.
7. Discard the accutase-supernatant and add calcium-free ringer solution.
8. Centrifuge at 1,100 rcf for 3min.
9. Discard the supernatant.
10. Resuspend the cells in L15 medium (Table 1).
11. Transfer the suspension to the poly-L-lysine
coated culture plates as described above (see 2.1.1, step 16).