HEK 293T Media:
500 mL Dulbecco’s Modified Eagle Medium (+ 4.5 g/L D-Glucose, + L-Glutamine,
+ 110 mg/L Sodium Pyruvate)
50 mL Fetal Bovine Serum (FBS or FCS) 5 mL Penicillin/Streptomycin
Ins-1 (832-3) Media:
1000 mL RPMI 1640 Medium (+ L-Glutamine)
2g Sodium Bicarbonate
100 mL Fetal Bovine Serum (FBS or FCS) 22 mL Ins-1 Supplement 10 mL HEPES Buffer 10 mL Penicillin/Streptomycin (if desired)
NIH3T3 Media—5% BCS, 5% NCS
For 1 liter: 900 mL DME (Dulbecco’s Modified Eagle Medium) 50 mL BCS (Bovine Calf Serum) 50 mL NCS (Newborn Calf Serum) 10 mL Pen/Strep
For 500 mL: 450 mL DME 25 mL BCS 25 mL NCS 10 mL Pen/Strep
For 450 mL: 400 mL DME 20 mL BCS 20 mL NCS 10 mL Pen/Strep
HEK 293T Freeze Media:
9 mL DMEM w/ 10% FBS 1 mL DMSO
For 1 Liter: 5 g. Glucose (powder) 10.6 g. F12 1.167 NaHCO3 ~950 mL ddH2O adjust pH to 7.15 q.s. to 1 L w/ ddH2O For 4 Liters: 20.0 g. Glucose (powder) 42.4 F12 4.7 g. NaHCO3 3800 mL ddH2O Adjust to pH 7.15 q.s. to 4 L.
DMEM for Tissue Culture:
*Make 16-20 L at a time.
For 4 L: 53.93 g. DME 14.8 g. NaHCO3 3800 mL ddH2O Adjust to pH 7.15 q.s. to 4 L. Filter sterilize, store at 4oC.
G418 Selection Media:
900 mL DME 100 mL FBS 10 mL Pen/Strep 15 mL G418 (want concentration to = 750 ug/mL)
G418 (Geneticin G-418 Sulfate) Stock Solution:
Want stock to be 50 mg/mL Add full bottle of G418 and 100 mL ddH2O Filter sterilize in hood in 150 mL Nalgene filter Dispense into 10 mL conical vials.
Polybrene (Hexadimethrine Bromide):
8 mg/mL (1000x) stock 80 mg in 10 mL ddH2O. Filter sterilize; store at -20oC.
7.5 % NCS + 2.5% FBS
750 mL NCS 25 mL FBS 10 mL Pen/Strep
1.0 mM Palmitate Media
- RPMI media (However much you want to make into palmitate media. Should be pH-ed, should have additives, but does NOT need to be sterile, since you’ll filter it later)
- CAUTION: Note that if you start with 500 mL plain RPMI media, it will actually be 571 mL after you add the additives. Keep that in mind for your calculations below.
- Ethyl Alcohol, Pure (use the smaller bottles, not the huge ones that are for sterilization)
- Sodium Palmitate (in fridge door)
- BSA (in fridge drawer)
- Hot Plate
- Stir Bar
- Small Glass Bottle (the ones with the orange, screw-on lid work great)
- Water Bath
- Medium Beaker
- Large Beaker
1. Fill large beaker with ~1 inch of water. Set up thermometer with a stand/clamp so that the tip is in the water but not touching the beaker, or else it can melt. Heat Large Beaker of water to 60-70˚C (ideally 65˚C) and set the hotplate to 90-100˚C once this temperature is reached to maintain it here. Check back often to make sure the temperature stays in this range or the ethanol you add later will boil, or the palmitate will coagulate.
2. Based on your desired end volume and concentration of palmitate media, calculate needed amounts of ethanol/palmitate mixture and ethanol and palmitate separately:
To make 1mM palmitate:
-1 mL of ethanol per 100 mL of media (ie. 5.71 mL ethanol for 571 mL of media)
-0.2784 mg of sodium palmitate per each mL of media (ie. 159 mg of sodium palmitate for 571 mL of media)
3. In a conical vial, measure out mg of palmitate and ethanol.
4. Cap tightly to ensure the ethanol doesn’t evaporate, and place container in the heated beaker.
While the Palmitate/Ethanol mixture is dissolving, prepare the BSA/media solution:
5. Add 2g of BSA per 100mL RPMI Media (with additives, ie. 11.24 g BSA for 571 mL media) and a stir bar and let stir until the palmitate is fully dissolved.
6. Check ethanol/palmitate to see if it is fully suspended. It won't fully dissolve but will appear as a viscous, white solution.
7. Once BSA is fully dissolved (media will no longer be cloudy, but instead appear clear, and no BSA clumps) place on the shaker plate at 70˚C.
8. Pipette the ethanol/palmitate solution into the media (it will be viscous so go slow).
9. Pipette media into the ethanol/palmitate vial and then back into the media to ensure all of the ethanol and palmitate is dissolved into the media.
10. Allow the media to mix on the shaker table for 30 minutes.
11. Sterile filter in the hood.
a. If making normal media simultaneously, filter the normal media first and then the BSA media last.
12. Your sterile Palmitate Media can now be stored in the Fridge, ready to use upon re-heating in water bath.
a. Be sure to label the container: *concentration* mM Palmitate Media, initials, and date