I. MEDIUM

A. S2, S2C, S2*, S2R+, S3, l(2)mbn, Kc(167), & DL2 cells:

Schneider's/ 10% FBS/ PS
450 ml Schneider's medium (Gibco #11720-034)
(pour off 50 ml into Falcon to save as serum-free)
***[S2R+ and Kc cells will not grow in Schneider's medium from Sigma]
50 ml Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 ml 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)

Sterile filter (0.2 µ) Store at 4^oC - do not freeze

B. ML-DmBG2 & ML-DmBG6 cells:

M3/10% FBS/PS/Insulin
450 ml Shields and Sang M3 insect medium (Sigma #S8523)
(pour off 50 ml into Falcon to save as serum-free)
50 ml Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 ml 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
10 ug/ml Insulin (Sigma #I6634)

Sterile filter (0.2 µ) Store at 4^oC - do not freeze

C. clone8 (CL8) cells:

Complete M3 Medium
Shields and Sang M3 insect medium (Sigma #S3652)
2% Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
2.5% Fly extract (see below)
0.0125 IU/ml Insulin (Sigma #I6634)
1x Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)

Sterile filter (0.2 µ)

Heat Inactivating Fetal Bovine Serum

1. Thaw the Fetal Bovine Serum on a shaker at 2-8oC (overnight)
2. Pre-heat a waterbath to a temperature of 56oC. (Make sure the water covers all of the serum in the bottle)
3. Heat the serum for 30 minutes in the 56oC bath.
4. Allow serum to cool to room temperature before adding to cells.

Insulin Stock (100x)
Dissolve 10mg (25IU) in 0.5ml of 0.01N HCL.
Heat in 37^oC to dissolve for few minutes.
Add 19.5mL of M3 Media to bring volume up to 20mL.
Filter Sterilize with a 0.22µm filter.
Alliquot and store stock at -20^oC. Working stock may be kept at 4^oC for 4-5 weeks.

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

S2*	3-4 days	1:3 - 1:4
S2c	3-4 days	1:3 - 1:4
Kc(167)	3-4 days	1:3 - 1:4
l(2)mbn	3-4 days	1:3 - 1:4

B. Adherent cell lines

DL2	3-4 days	1:8 (1:5 - 1:10)
S3	3-4 days	1:3
S2R+	3-4 days	1:3 - 1:4
CL8	4-5 days	1:5-1:10
BG2	4-5 days	1:3-1:4
BG6	4-5 days	1:3-1:4

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

Protocol 2:
Remove medium
Wash in PBS to remove any serum
Add 5 ml trypsin; let sit 5-10'
Bang cells off flask.
Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
Spin down cells at 1200-1400 rpm for 5 min.
Resuspend cells in serum medium.
Aliquot cells into new flasks accordingly

Protocol 3:
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

BG2

Remove medium (save and filter through syringe filter for use as "conditioned medium").
Wash in PBS to remove any serum
Add 5 ml trypsin; let sit 5-10'
Bang cells off flask.
Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
Spin down cells at 1200-1400 rpm for 5 min.
Resuspend cells in conditioned medium.
Aliquot cells into new flasks accordingly

Can also use Accutase instead of trypsin.

IV. FREEZING

Grow cells to subconfluencey - approximately 1-2 x 10⁷ cells/ml.
Label appropriate # of cryovials.
Remove cells from flask (trypsinize and resuspend in medium if necessary).
Count cells.
Spin cells at 1200 rpm 5'
Aspirate off medium.
Resuspend at approximately 1.1 x 10⁷ cells/ml in freezing medium:
  FBS + 10% DMSO, filtered OR Complete medium + 10% DMSO, filtered
Aliquot 1ml cell suspension/cryovial.
Put vials in foam box, tape closed, and place in -70^oC. After a few days, transfer vials to LN₂ for long term storage.

V. THAWING

Prepare 15ml conical tube with 5ml medium.
Thaw cells quickly in water bath.
Just before cells are completely thawed, decontamiate the outside of the tube with 70% EtOH.
Transfer the cells to the conical tube with 5ml medium.
Spin at 1200rpm 5'.
Aspirate and resuspend cells in 5ml medium.
Plate in a T25 flask. Watch daily. Initially, cells may need to be split at irregular intervals.

VI. COUNTING

Remove ~0.5 ml cells from flask (trypsinize cells if necessary) into eppendorf.
Dilute cells into Trypan Blue according to estimated density.
(confluent cultures try 30:70 µl to 50:50 µl of cells:TB).
Transfer 10 - 12 µl cells to haemocytometer.
Count cells in opposite corners (~100-200/ large square).
Calcuation:
    (count #1 + count #2) / 2 x (dilution factor) x 10⁴ = X cells/ml (usually ~1-10 x 10⁷)