Methods
Special Tissue Culture Media
T–Medium (500 ml)
(For growing C4-2B cells)
Keller Lab
(June Escara-Wilke, corrected 7/13/12)
Can use InVitrogen Formula # 02-0056DJ (add 10% FBS, 1% Pen Strep)
Complete T-medium contains per 500 ml (make 2 bottles)
75 ml of F12-Ham (Cat# 11765-054)
375 ml of DMEM low glucose (InVitrogen Cat #11885-084)
1 ml of insulin (2.5 mg/ml stock soln.=500X)
1 ml of T3 (triiodo-thyronine, 6.825ng/ml stock soln in 0.1%BSA in PBS.=500X)
1 ml of apo-transferin (2.5mg/ml =500X)
1 ml of d-Biotin (1.22mg/10ml 0.1%BSA in PBS + NaOH =500X)
2 ml of Adenine Hemi-Sulfate (12.5 mg/ml in 0.1% BSA in PBS + NaOH)
5 ml of Pen-Strep (2 x 5 ml aliquots)
50 ml FBS (2 x 50 ml aliquots)
---Measure the volume of DMEM using cleaned-for-TC graduated cylinder. Pour medium in the funnel portion of the 500 ml filter unit (PES membrane, 0.22um). Add and mix the supplements to the medium. Filter sterilize inside the TC hood.
To make the supplements:
1. Insulin (from bovine pancreas, Sigma I5500-500mg)
Dissolve 50 mg of insulin in 12.5ml of acid solution (Add 40ul of 2.5N HCl to 12.5 ml distilled water), stir until dissolved (make sure it is completely dissolved –no precipitates falling off of solution. Add more acid dropwise if necessary)
Add 5.5 ml of ddH2O to above, pipette up and down.
Add 2.0 ml MEM, then pipette up and down. Total of 20 ml.
Sterile filter in the hood using 0.22um syringe filter and 30 ml syringe. Concentration = 2.5 mg/ml working stock
Make 1 ml aliquots in sterile 1.7 ml microfuge tubes. Store in -20C freezer.
Add 1 ml per 500 ml of T-medium
2. T3 (3’3’, 5-triiodo-L-thyronine, Sigma T2752-100mg)
Dissolve 10mg (0.01g) of T3 in 10ml of 0.1%BSA PBS (=1mg/ml stock), add several drops of 1N NaOH to help dissolve. Concentration: 1 mg/ml T3 = Storage stock.
Add 10 ul of 1mg/ml T3 to 10ml of 0.1%BSA PBS (=1ug/ml)
Finally, add 68.25ul of 1ug/ml T3 to 10ml 0.1%BSA PBS
Make 1 ml aliquots in sterile 1.7 ml microfuge tubes
Add 1 ml per 500 ml of T-medium
3. apo-Transferrin (human, Sigma T2036-100mg or T-4382-1G)
Add 0.025g or 25mg of Apo-transferin in 10ml of 0.1%BSA PBS(2.5 mg/ml), vortex; add 1 to 2 drops of 1N NaOH to help dissolve (if not dissolved).
Make 1 ml aliquots in sterile 1.7 ml microfuge tubes
Add 1 ml per 500 ml of T-medium
4. d-Biotin (Sigma (Sigma B-4501 1g)
Dissolve 0.1g d-Biotin in 10ml of 0.1%BSA PBS (=10mg/ml stock), add several drops of 1N NaOH to help dissolve
Add 122 ul of 10mg/ml d-Biotin to 10ml of 0.1%BSA PBS
Make 1 ml aliquots in sterile microfuge tubes
Add 1 ml per 500 ml of T-medium
5. Adenine Hemi-Sulfate (Sigma A3159-5G)
Dissolve 0.1563g of Adenine Hemi-Sulfate in 25ml of 0.1% BSA in PBS, add several drops of 1N NaOH to help dissolve.
Make 1 ml aliquots in sterile microfuge tubes (62.5mg/ml)
Add 2 ml per 500 ml of T-medium (12.5 mg/ml total)
MDA PCa 2b Medium (500 ml)
(For growing PCa 2b cells)
Keller Lab
(June Escara-Wilke, formulated 11/7/18)
Use ATCC 30-2004 F12K Medium or GIBCO F12K
500 ml of F12K Medium (Cat# )
~20% non heat-inactivated Fetal Bovine Serum = 90ml
5.9ml of GIBCO Pen-Strep Solution 100x
0.5 ml of Cholera Toxin (25 ug/ml working stock=1000X) = Final 25ng/ml CT
0.5 ml of mouse Epidermal Growth Factor (10ug/ml working stock=1000X) = Final 10ng/ml
0.5 ml of Phosphoethanolamine MW=141.06 (5mM working =1000X) = Final 0.005mM or 5uM
0.5ml of Hydrocortisone ( 0.1ug/ml working stock= 1000X) = Final 100pg/ml or 0.1ng/ml
0.5ml Sodium Selenite FW=172.9 (45uM working stock (1000X) =Final 45nM
0.5ml human recombinant insulin. Received 100mg/ml stock. Prepare final 0.005mg/ml or 5ug/ml
Pipet the supplement aliquots directly to the F12K bottle, rinsing the tube once with medium. Mix supplement with medium.
Transfer medium to a 500 ml filter unit (PES membrane, 0.22um). Filter sterilize inside the TC hood.
To make the supplements:
1. Cholera Toxin ( Sigma C8052 = 1mg vial) Hazard: Poison. Read SDS before use when making stock solutions Wear complete PPE face shield, googles, disposable gown, nitrile gloves.
Dissolve 1 mg of CT in 1ml of sterile GIBCO H2O by injecting with a syringe = 1mg/ml. Swirl vial until dissolved or pipette up and down. Avoid making bubbles.
Place 1.5ml of GIBCO water in a 50 ml tube. Add 0.5ml of the 1mg/ml stock (1:4 dilution = 250ug/ml.
Dilute 1:10 by adding 18ml of water = 25ug/ml working stock
Sterile filter in the hood using 0.22um 50ml Steriflip or a 0.22um filter attached to a 20 to 30ml syringe.
Make 0.5 ml aliquots in sterile 1.7 ml microfuge tubes. Store in -20C freezer.
Add 0.5 ml per 500 ml of F12K medium
2. mouse Epidermal Growth Factor (Corning Cat # 354010)
Dissolve 1mg mEGF in 100ul of 10mM HAc, mix till dissolved, then add 0.9ml H2O =1mg/ml storage stock.
Add 100 ul of 1mg/ml mEGF to 10ml of 0.1%BSA PBS (=10ug/ml)
Make 0.5 ml aliquots in sterile 1.7 ml microfuge tubes
Add 0.5 ml per 500 ml of F12K medium. Store aliquots in -20C.
3. Phosphoethanolamine (Sigma Cat # P0503) Hazard: Extreme danger- causes severe skin burns and eye damage; do not breathe dust or mist; wear face shield, googles, masks, disposable gown when measuring from powder
Add 1g of PE to 10 ml of water, vortex = 100mg/ml
To make 5mM working stock (=0.705mg/ml), take out 70ul of the 100mg/ml storage stock and add to 10ml of water = 5mM (1000X)
Make 0.5 ml aliquots in sterile 1.7 ml microfuge tubes
Add 0.5 ml per 500 ml of F12K medium. Store aliquots in -20C.
4. Hydrocortisone: 1mg/ml from Shamileh
Take 10ul and add to 10ml GIBCO H2O (1:1000 dilution) =1ug/ml
Take 1 ml 1ug/ml and add 9ml of GIBCO H2O (1:10 dilution) = 0.1ug/ml (1000X)
Make 0.5 ml aliquots in sterile 1.7 ml tubes and store in -20C freezer.
Add 0.5 ml per 500 ml of F12K.
5. Sodium Selenite Sigma S5261 FW = 172.94 or 173
Dissolve 7.8 mg of Sodium Selenite in 1 ml of GIBCO H2O = 45mM stock
Add 10ul of 45mM stock to 10ml of GIBCO H2O (1:1000dilution) = 45 uM working stock
Make 0.5 ml aliquots in sterile microfuge tubes and store in -20C.
Add 0.5 ml per 500 ml of F12K medium (Final 45nM)
6. Human recombinant Insulin (Life Technologies cat # 12585-014) Received 50ul 100mg/ml
Dilute 1:20 to 5mg/ml: 10 ul to 200ul total volume with GIBCO H2O, or 50ul 100mg/ml added to 1ml (after removing 50ul) = 5mg/ml
Make 0.5 ml aliquots in sterile 1.7 ml tubes and store at -20C.
Add 0.5ml per 500ml of F12K (final 5ug/ml)