2.1.1. Protocol for pupal cells

For the isolation of neuronal cells from pupae:

1. Collect 13-14 day old pupae (red-eyed pupae, see BEEBOOK paper on miscellaneous research methods (Human et al., 2013) for the method to obtain them).

2. Remove pupae carefully from brood cell with forceps. Make sure that the head is not even slightly turned and that the neck is not stretched.

3. Separate the head from thorax by means of a scalpel and pin it down on a petri dish (35 mm in diameter) with wax, paraffin or silicon coated dish with micro pins near the antennae.

4. Make a cut axial around the head by means of a scissor. Start on one mandible, go over the backside with the developing ocelles and finish on the second mandible. Make sure to cut not only the cuticula of the pupae but also the head capsule underneath.

5 .Cover the head with L15 medium (Table 1).

6. Remove the complete head capsule carefully without the brain.

7. Separate the brain from the head.

8. Remove the visible neurons (optical lobes) from the brain as well as the ocelles and the eye-retina, otherwise growing of neurons will be suppressed.

9. Prepare a 24-well-plate (sterile tissue culture quality) with fresh cold (4°C – 10°C) L15 medium.

10. Choose the parts of the brain that will be used for cell culture and transfer them to a medium-filled well of the 24-well plate (see step 9).

11. Prepare as many brains as needed (a minimum of five is recommended to obtain enough cells) following the above outlined procedure.

12. After collecting enough brains, transfer them to a 1.5 ml-reaction tube with calcium-free ringer solution (Table 1).

13. Incubate the brains for 10 min in the ringer solution.

14. Aspirate the ringer solution and add cold L15 medium (for 5 brains add 1000 µl L15).

15. Carefully resuspend the brains with a pipette (1 ml pipette tip) in the L15 medium to disintegrate the tissue.

16. Transfer the cell suspension to a poly-L-lysine coated cell culture plate (10cm2, commercially available from several suppliers).

17. Let the cells attach for 20 min.

18. Carefully add 4 volumes of pre-warmed (27°C) BM3 medium   (Table1) supplemented with 10 % antibiotic/antimycotic solution, pH 6.7 (Sigma-Aldrich, A5955).

19. Cultivate the cells at 27°C in an incubator suitable for insect cell culture [cooling incubator]; avoid desiccation of the cells by placing water filled bowls into the incubator.

20. If the medium becomes viscous, change the BM3 medium after a week.

The cells are vital for a minimum of 14 days.

Table 1. Recipes for media used for cultivation of primary neuronal and gut honey bee cells as well as non-permanent honey bee cell lines.

L 15 medium, pH 7.2

14.9 g L-15 powder, 4.0 g glucose, 2.5 g fructose,
3.3 g prolin, 30 g sucrose, dissolve in bi-distilled water and fill-up to 1000 ml with bi-distilled water; adjust pH 7.2 with NaOH

BM 3 medium, pH 6.7

1000 ml L 15 medium, 0.75 g Pipes, 30 ml FCS (heat inactivated), 12 g Yeastolate

AmWH5 medium (Hunter, 2010)

500 ml Grace’s insect medium (supplemented), 500 ml Schneider’s insect medium, 1000 ml 0.06 M L-histidine hydrochloride monohydrate (pH 6.5), 20 ml M199 medium (10X) with Hank’s salts, 34 ml medium CMRL 1066, 66 ml Hank’s balanced salts (1X), 52 ml 2 N glucose solution (filter sterilized, adjust osmolarity), 108 ml foetal bovine serum (FBS, heat inactivated)

Add to final volume of medium (2280 ml):
3 ml L-glutamine (100X), 3 ml MEM (50X) amino acid solution, 3 ml gentamycin (10,000 U/ml), 5 ml PenStrep (100X)

Note: 0.05 M HEPES buffer (pH 6.5) works as substitute for L-histidine monohydrate; final osmolarity is about 380 mOsm/l; can use Grace’s insect medium as primary medium, with no Schneider’s medium

ringer solution, Ca-free,  pH 7.2

(147 mM NaCl, 5 mM KCl, 65 mM HEPES)

8.6 g NaCl, 0.36 g KCl, 15.6 g HEPES; dissolve in bi-distilled water and fill-up to 1000 ml with bi-distilled water; adjust pH 7.2 with NaOH

1 X PBS, pH 7.4

(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4)

8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4;  dissolve in bi-distilled water and fill-up to 1000 ml with bi-distilled water; pH will be between 7.2-7.6