Cell Culture Media

HEK 293T Media:Whiskers

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:

500 mL RPMI 1640 Medium (+ L-Glutamine)
50 mL Fetal Bovine Serum (FBS or FCS) 11 mL Ins-1 Supplement 5 mL HEPES Buffer 5 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

 

F-12 Media

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)
     - Foil
     - Water Bath
     - Medium Beaker
     - Large Beaker
     - Thermometer
     - Scale

Procedure:
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 0.15mM Palmitate Media:
- 60ul of Ethanol/Palmitate mix per 10mL BSA media
To make 1 mM Palmitate Media:
- 400ul of Ethanol/Palmitate Solution per 10mL BSA Media
For other concentrations, where x is desired concentration:
- x/0.15=y
- y*60ul=z
- z ul of Ethanol/Palmitate Solution per 10mL of BSA Media 
To make V desired total mLs of palmitate media:
- V/10mL=t
- z*t= m ul (total amount of Ethanol/Palmitate you need to add to you BSA media)
- m+some extra = amount of ethanol you’ll add to your palmitate
20mL Ethanol needs 139.205mg Palmitate, so 
- (m+some extra)/20))*139.205= p = amount of Palmitate needed for m ml ethanol 
Ex.: If I wanted to make 300 mL of normal RPMI media into 100 mM palmitate media, then I need 400ul of Ethanol/Palmitate Solution per 10mL BSA Media, so t = 300/10 = 30 (300 mls is 30 ten-ml. samples), so m = 400*30 = 12,000 ul, so I would need to add a total of 12 mls ethanol/palmitate solution to my 300 mL of normal media. So m = 12, but to make sure I have enough I’ll make 15 mls (m+some extra) of ethanol/palmitate mixture. Which means I need to use .75*139.205 = 104.4 mg of palmitate.

3.  In the small bottle, measure out p mg of palmitate. Blow the extra palmitate off of the rim to ensure a more exact measurement.
4.  Add m+some extra mL of Ethyl Alcohol to the bottle, pipetting slowly all along the circumference just under the rim to ensure all Palmitate gets in solution.
5.  Add a mini stir bar to the solution, cap the bottle tightly to ensure the ethanol doesn’t evaporate, and place container in the heated beaker.
6.  Turn the stir plate on full and position the container in such a way that the stir bar bounces around the container in order to break up the palmitate chunks.

While the Palmitate/Ethanol mixture is dissolving, prepare the BSA/media solution:
7.  To make 2% BSA media (you need the same volume (V) of this as you ultimately want of palmitate media), add 2g of BSA per 100mL RPMI Media (with additives) and stir with a flat-headed spatula/scooper. Try to get BSA clumps off the sides of the beaker and into the solution.
8.  Once stirred as well as possible, cover beaker and spatula with tin foil and place in a water Bath so that the BSA completely dissolves.
9.  Check ethanol/palmitate to see if it is fully dissolved. Turn the bottle in your hand so that the solution covers each side of the container to make sure that all the palmitate is in solution.
a.  Keep the solution in the beaker of water, maintaining it at a temperature of 65˚C until ready to add it to the BSA media solution.
b.  The solution should be clear. If it is cloudy, the palmitate is coming out of solution and should be reheated.
10.  Once BSA is fully dissolved (media will no longer be cloudy, but instead appear clear, and no BSA clumps), filter the BSA media into sterile bottle in the hood.
a.  If making normal media simultaneously, filter the normal media first and then the BSA media last.
11.  Add the needed amount (m) of the Palmitate/Ethanol Solution to the purified BSA media in the hood. Be quick so that the palmitate doesn’t come out of solution and so that the ethanol doesn’t leak out of the pipette.
- don’t worry, because it’s ethanol, this is already sterile without filtering
12.  Immediately mix thoroughly, so as to incorporate the BSA media with the Palmitate/ethanol solution.
13.  Clean the container containing the extra palmitate/ethanol solution immediately so that it doesn’t form a sticky gel that is hard to clean.
14.  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