Fly Extract

1. Collect about 30g of healthy flies.
   200 flies weigh about 0.22g, use 1.5 ml M3 medium for 0.22g.
   To make 200 ml homogenate, use 30g of flies. (7.5ml/ 1g).
2. Place flies in freezer for ~20'. Remove flies from freezer and weigh out 1/2 of them and put rest back in freezer. Add the appropriate amount of M3 medium.
3. Place flies plus medium in a blender. Blend at medium speed until it looks as if all the flies have been lysed and the supernate is reddish from the eye pigments.
   [Old method: Remove 5ml of the fly suspension and crush in a dounce homogenizer (which should be extremely clean) until the plunger reaches the bottom of the homogenizer. Collect the resultant mush in 50ml conical tubes. Repeat until all flies are homogenized.]
4. Transfer lysate to 50ml conical tubes and spin at 2600 rpm in a tabletop centrifuge at RT. Remove the supernate and transfer to a new tube.
5. Remove the oily top layer.
6. Heat inactivate the extract in a 60^oC waterbath for 10'. You will see a precipitate form.
7. Centrifuge at 2600 rpm for 1 hour at RT. Remove supernate and sterilize through 0.22 µm filter. You will go through several filters, so either use a prefilter, or be prepared to waste a lot of prepackaged filters.
8. Store in 12.5 ml aliquots (2.5% final in 500ml) and keep at -20^oC after freezing in LN₂.

II. GROWTH CONDITIONS

Cells grow @ 23-25^oC and @ RT
Do not need CO²
Split every 3-4 days to maintain
Density sensitive - die if too dense or too dilute

III. MAINTENANCE

Split one flask of cells (most recent date) into two new T75 flasks (VWR# BD353136) when culture is confluent. Keep other flask as a backup. Monitor growth status by microscopy before splitting and decide whether to adjust recommended dilution factor accordingly.

A. Semi-adherent cell lines - will stick to new flask but come loose over time

B. Adherent cell lines

SL2, S2C, S2*

Detach cells from the flask either by banging or scraping
Pipet cells up and down about 10 times to resuspend cells and separate clumps
Aliquot cells into new flasks accordingly

DL2, DL1, S2R+, Kc167, Clone 8, S3

Protocol 1:
Remove all medium
Scrape cells with scraper
Resuspend cells with 10mL of fresh medium, pipetting up and down about 10 times
Aliquot cells into new flasks accordingly

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