|Methods in this section:|
|Fly extract 2 for routine culture (FE2) |
1. First collect your flies; tip them from culture bottles into an empty glass bottle and plug it. Put it in the freezer for 45 mins or so until the flies are quiescent (probably dead). This can be done in advance, and flies stored in the freezer, if you want to collect a nice big batch of flies.
|2. Set the centrifuge to cool to 4 deg.C. |
3. Have some ice ready in a polystyrene box.
4.Weigh a small sterile plastic pot. When the flies are ready, tip them into the pot and reweigh it. About 200 flies weigh 0.22g, and need 1.5 ml SS3 (unsupplemented) for squashing.
5. Tip a few ml medium into the glass homogenising tube and add about 1 teaspoon flies. Squash until the homogeniser plunger reaches the bottom of the tube. Tip the resulting fly porridge into a centrifuge tube and keep on ice. If the enzyme tyrosinase acts it goes black and is useless. The porridge is a tasteless salmon orange colour with black and brown bits of exoskeleton. Carry on crushing until all flies and medium are used up. Nearly fill the centrifuge tubes.
The fly porridge looks like this:
|6. Spin the tubes of fly porridge at 2600rpm (1500 g) at 4° C for 15 mins. Switch on the 60°C waterbath. |
After the first spin the extract looks like this:
7. Remove supernatant, including surface fat/oil, from the spun tubes with a pasteur pipette and put into fresh tubes. Discard the exoskeleton pellet and the terracotta-coloured layer of eye bits.
8. Put the tubes in the 60°C waterbath for 5 mins to inactivate and precipitate enzymes.
|9. Spin at 2600rpm (1500g) at 4° C for 90mins. |
After this spin the tubes look like this:
10. Remove the supernatant and pool it in one container. Filter-sterilise the extract through 0.22µm filters. If the extract is still cloudy you may have to filter through 0.8µm and 0.45µm filters first. Aliquot to 2.5ml and freeze at -20°C.
|Fly Extract 1 for initiating primary culture (FE1) |
1. Procedure as for FE2 but the flies must be at least 75% female. So, collect and freeze flies as above.
2. Tip a spoonful of flies into a glass Petri dish or similar, and sort out 150 nice fat females. Put them in a bijou bottle or other small container. Then add another fifty flies without sorting them. If they look like waking up you can put your sorting dish and containers on a bed of ice.
3. When you have done as many as you can bear, put two lots, i.e. 400 flies, into the homogeniser and squash with 3ml SS3. Tip this into a centrifuge tube and keep on ice. Squash the rest of the flies in similar fashion and add to the centrifuge tube.
4. From now on the procedure is the same as for FE2, except we find 150µl frozen aliquots more convenient.
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