This is our lab protocol for growing imaginal disc cells (clone-8)
The Milner Lab website has additional protocols for clone-8 cells.
Clone 8 cells are passaged every 3-4 days from confluent T25 flasks (about 5 x 107 cells): split roughly 1/5 to 1/10. Never allow cultures to become too dense for they will die rapidly! If 95% are dead the day after you split them you have probably let them grow too dense. For the same reason, always use subconfluent cells for biochemistry or RNA isolations. These cells are tightly adherent, especially when they have been passaged recently. Wash the cells with 3ml of trypsin, prior to trypsinization. Trypsinize for about 1 min at room temperature. Tap the flasks real hard to detach cells, immediately add 5 ml of medium to inactivate the trypsin. Spin down cells at 1200rpm in a table top centrifuge and resuspend to required density. Always use a fresh flask when transferring cells.
Cells can be frozen in complete M3 medium + 10% FCS and 10% DMSO; again use subconfluent cultures.
Place flies in freezer for 20 min.
Place 30g of frozen flies and 200ml of medium into a blender.
"Puree" in blender approximately 2-3 minutes.
Spin mush at 3000 rpm in a table top centrifuge for 20 min.
Remove supernatant (leaving exoskeletons and eye pigment) and transfer to a new tube.
Remove oily layer on top.
Heat inactivate in 60 C waterbath for 30 min. You will see a precipitate form.
Centrifuge at 3000 rpm for 30 min.
Transfer supernatant to new tube and spin again for 15 min.
Remove supernatant and sterilize through a 0.22 um filter.
Store in 12.5 ml aliquots (2.5% final in 500ml) and keep at -20 C.
2.5% fly extract
5 ug/ml insulin
1/2 x penstrep (100 x bottle = 5000 ug/ml)