TITLE: Making E8 medium for ES/iPS culture
AUTHORS: Jiuchun Zhang, Harper JW

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about making E8 medium for ES/iPS culture.</div></div>

[STEPS]
?. [Preparation of Recombinant Human TGF-beta 1 ]
**Most temperature sensitive, work fast***This is for large number of aliquots. Change volume according to your own scale.
Order 1 mg but have them provide it in 2 x 500 μg aliquots plus a sample for testing if theywill do this. 1000X = 1.745 μg/mLFor 500 μg protein: bring to a total volume of 286.5 mL in bufferUse   containing   Chilled Buffer (make about 300-500 mL of buffer)
?. [Preparation of Recombinant Human TGF-beta 1 ]
Prepare buffer:For 100 mL: (in fume hood cabinet) for final concentration of Add  for final  concentrationFor 500 mL:  +  + This can be made at any point and stored at .
[HCl (11.6M)]
[HSA (50 mg/mL)]
[HCl]
[HSA ]
[H2O]
4 °C
?. [Preparation of Recombinant Human TGF-beta 1 ]
Bring Solution to  for  – do not need to do this if buffer already cold.
4 °C
on ice
?. [Preparation of Recombinant Human TGF-beta 1 ]
Set up tubes in hood – will need 2 hoods (over 500 aliquots).
?. [Preparation of Recombinant Human TGF-beta 1 ]
To a 250 mL Millipore filter, add .
[chilled buffer]
?. [Preparation of Recombinant Human TGF-beta 1 ]
Then add your  (note the total volume – use pipet to measure) – thaw , will take around - .
[bottle of TGFb from R&D Systems]
on ice
?. [Preparation of Recombinant Human TGF-beta 1 ]
For TGFbeta coming in the form of lyophilized powder, dissolve in the chilled buffer at , then add to appropriate amount of buffer.
?. [Preparation of Recombinant Human TGF-beta 1 ]
Bring to a total volume of 286.5 mL.
?. [Preparation of Recombinant Human TGF-beta 1 ]
Filter.
?. [Preparation of Recombinant Human TGF-beta 1 ]
Aliquot /tube (get some help because that’s over 500 aliquots and you want to work quickly to get them frozen).
500 µl
?. [Preparation of Recombinant Human TGF-beta 1 ]
Label.
?. [Preparation of Recombinant Human TGF-beta 1 ]
Freeze @  for immediate use or  for long term storage.
-20 °C
-80 °C
?. [Preparation of Holo-Transferrin ]
1000X = 10.67 mg/mL à 426.8 mg (+/- 5mg) / 40 mlUse chilled PBS to dissolve this
Change scale accordingly if you are making a different amount.
?. [Preparation of Holo-Transferrin ]
Static zap your Falcon Tube and weigh paper before transferring the crystals.
?. [Preparation of Holo-Transferrin ]
On weigh paper, measure out .
[Holo-Transferrin]
?. [Preparation of Holo-Transferrin ]
Add your .
[chilled PBS]
?. [Preparation of Holo-Transferrin ]
Vortex gently until in solution, avoid foaming.
?. [Preparation of Holo-Transferrin ]
Filter with Steriflip.
?. [Preparation of Holo-Transferrin ]
Aliquot / tube.
500 µl
?. [Preparation of Holo-Transferrin ]
Label.
?. [Preparation of Holo-Transferrin ]
Parafilm bottle of holo-transferrin when done.
The solution will be red in color.If you freeze it at   it will be orange in colorIf you freeze it at   it will turn clearIf you freeze it at   and move it to   it will slowly turn from orange to clearThe color is an indicator of the oxidation state, either way it’s all right to use
?. [Making E8 medium for ES/iPS culture]
Dissolve  in . Warm the water at  to completely dissolve the salt.
[sodium bicarbonate]
[water]
37 °C
?. [Making E8 medium for ES/iPS culture]
Dissolve  in . Then dilute this solution 1:100 with water to make  solution.
[sodium selenite]
[water]
?. [Making E8 medium for ES/iPS culture]
Add  to the . Then filter sterilize.
[100 μg/ml sodium selenite solution]
[sodium bicarbonate solution]
?. [Making E8 medium for ES/iPS culture]
Aliquot into 10 ml aliquots. Each aliquot is enough for 500 ml of DMEM/F12.
?. [Making E8 medium for ES/iPS culture]
Dissolve  () in . Filter sterilize. Then aliquot into 1 ml aliquot. Each aliquot is enough for .
[L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate]
[water]
[DMEM/F12]
?. [Making E8 medium for ES/iPS culture]
Freeze all aliquots at .
-80 °C
?. [Making E8 medium for ES/iPS culture]
To make E4 media. Add  and  from above to .
[sodium bicarbonate and sodium selenite solution]
[VitC solution]
[DMEM/F12]
?. [Making E8 medium for ES/iPS culture]
E4 media made this way should have osmolarity around 310 and pH around .
?. [Making E8 medium for ES/iPS culture]
To make E6 media, add to the above E4 medium the following:  and . Mix well.
[insulin solution (10 mg/ml)]
[transferrin (10 mg/ml)]
(Note: The original E8 formula contains  . This recipe contains  . Both should work well for ES/iPS culture).
?. [Making E8 medium for ES/iPS culture]
To make complete media (E8), add to the above E4 medium the following:, , , . Mix well. Thaw all additives on cold-rack . Add cold base-media and wait until it is thawed, then add.
[insulin solution (10 mg/ml)]
[FGF2 (100 μg/ml)]
[TGFb1 (1.7 μg/ml)]
[transferrin (10 mg/ml)]
on ice
?. [Making E8 medium for ES/iPS culture]
The complete medium is good for 2 weeks at . DO NOT warm the media before feeding. Warming up the media will destabilize the growth factors in the media.
4 °C
?. [Making E8 medium for ES/iPS culture]
E4 and E6 media is good for at least 3 months at , possibly up to a year.
4 °C