100x Stock: 10 mg/ml in water. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C.
Insulin
Sigma
I-6634
200x Stock: 10 mg/ml in 0.05N HCl. Filter through 0.2 um syringe filter (cellulose acetate). Store in 120 ul aliquots (for 20 ml DMEM) at -20C.
Putrescine
Sigma
P-5780
100x Stock: 10 mM in ddH2O. Filter through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20 C
Selenium
Sigma
S-5261
100x Stock: 3 uM in ddH2O. Put 0.0051 g Selenium in a 15 ml tube labeled A (3 mM stock). Add 10 ml of sterile water. Take 10 ul from Tube A to Tube B with 10 ml ddH2O (3 uM stock). Filter Tube B through 0.2 um syringe filter (cellulose acetate). Store in 220 ul aliquots (for 20 ml DMEM) at -20C
Progesterone
Sigma
P-6149
100x Stock: 2 ug/ml
1. Add 1ml of 100% EtOH to 0.001 g Progesterone bottle
2. Add 49 ml ddH2O
3. Transfer 1 ml of this to second tube with 9 ml ddH2O
4. Filter through 0.2 um syringe filter (cellulose acetate)
5. Store in 220 ul aliquots (for 20 ml DMEM) at -20C
DDM1
Medium
To 10 mls of DMEM add from stocks:
100 ul Transferrin,
100 ul Putrescine,
100 ul Selenium,
100 ul Progesterone,
50 ul Insulin
Petri dishes
Cover-slips
Bellco Biological Glassware
1943-00012
Low lead glass, autoclaved.
Paper filter
Whatman
#1
10cc syringe
Tool
The cultures made in this media must be maintained in a 5% CO2 incubator at about 22-24°C. We use a standard mammalian tissue culture incubator placed in a cold room and heated to 23°C. Always used autoclaved filtered water and make in containers that are reserved for media and supplements only. Never put a pH meter or stir bar used for other purposes in media.
Sicaeros, B., O’Dowd, D. K. Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos. J. Vis. Exp. (5), e226, doi:10.3791/226 (2007).