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:

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.

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