Making E8 medium for ES/iPS culture

Prepare buffer:

For 100 mL:

1. 34.4µL (in fume hood cabinet) for final concentration of 4millimolar (mM)
2. Add 2mL for final 1mg/mL concentration
3. Bring vol up to 100 mL with molecular grade water

For 500 mL: 172µL + 10mL + 489.83mL

This can be made at any point and stored at 4°C.

Bring Solution to 4°C 4On ice for 0h 30m 0s – do not need to do this if buffer already cold.

Set up tubes in hood – will need 2 hoods (over 500 aliquots).

To a 250 mL Millipore filter, add 100mL.

Then add your 500µg (note the total volume – use pipet to measure) – thaw 4On ice, will take around 0h 30m 0s-0h 45m 0s .

For TGFbeta coming in the form of lyophilized powder, dissolve in the chilled buffer at 100μg/ml, then add to appropriate amount of buffer.

Bring to a total volume of 286.5 mL.

Filter.

Aliquot 500µL/tube (get some help because that’s over 500 aliquots and you want to work quickly to get them frozen).

Label.

Freeze @ -20°C for immediate use or -80°C for long term storage.

Static zap your Falcon Tube and weigh paper before transferring the crystals.

On weigh paper, measure out 426.8mg.

Add your 40mL.

Vortex gently until in solution, avoid foaming.

Filter with Steriflip.

Aliquot 500µL/ tube.

Label.

Parafilm bottle of holo-transferrin when done.

Dissolve 1.358g in 50mL. Warm the water at 37°C to completely dissolve the salt.

Dissolve 10mg in 1mL. Then dilute this solution 1:100 with water to make 100μg/ml solution.

Add 350µL to the 50mL. Then filter sterilize.

Aliquot into 10 ml aliquots. Each aliquot is enough for 500 ml of DMEM/F12.

Dissolve 160mg () in 5mL. Filter sterilize. Then aliquot into 1 ml aliquot. Each aliquot is enough for 500mL.

Freeze all aliquots at -80°C.

To make E4 media. Add 10mL and 1mL from above to 500mL.

E4 media made this way should have osmolarity around 310 and pH around 7.4.

To make E6 media, add to the above E4 medium the following: 0.25mL and 0.5mL. Mix well.

To make complete media (E8), add to the above E4 medium the following:

25mL, 0.5mL, 0.5mL, 0.5mL. Mix well. Thaw all additives on cold-rack -80On ice. Add cold base-media and wait until it is thawed, then add.

The complete medium is good for 2 weeks at 4°C. DO NOT warm the media before feeding. Warming up the media will destabilize the growth factors in the media.

E4 and E6 media is good for at least 3 months at 4°C, possibly up to a year.