B8 consists of a 1-liter bottle of DMEM/F12 (Corning) and a 2 mL supplement aliquot.
We standardly used the 'low-cost, easier to make' variant of B8 from Fig. S5C (Kuo et al., 2020).
Our recent Current Protocols in Stem Cell Biology protocol is available here. Note, we now use a larger 2 mL aliquot as 1.5 mL aliquots can show some solubility issues. No effect on cell growth was noted.
It should take less than 1 hour to make 100 aliquots of supplement, including aliquoting time.
Single supplement aliquot
Place 35 mL of sterile Milli-Q water and 7 mL of HCl into a 50 mL Falcon tube. Add 20 g of L-ascorbic acid 2-phosphate, mix vigorously and place on a rocker at 50 rpm for 10 min or until dissolved. Should be pH 7.1.
Place 48 mL of sterile Milli-Q water into a 50 mL Falcon tube. Add 0.5 g of insulin, add 400 µL of HCl and mix by inversion until dissolved (~ 1 min). Add 550 µL of NaOH (to pH 7.1), mix by inversion until dissolved.
Place 47 mL of sterile Milli-Q water into a 50 mL Falcon tube. Add 0.5 g of Optiferrin and mix by inversion until dissolved, add 1 mL of 2 mg/mL sodium selenite, 1 mL of 4 mg/mL FGF2, 40 µL of 250 µg/mL TGFB3, and 100 µL of 100 µg mL NRG1, mix until dissolved.
Add three tubes along with 50 ml of sterile Milli-Q water to a sterile bottle and mix. Run the entire 200 mL solution through a 250 mL bottle-top PES filter to sterilize, make 100 x 2 mL aliquots in sterile 2 mL Eppendorf tubes. Freeze at -20 °C
Costs to make enough supplement for 100 liters
Prices updated July 2020
All prices are the actual prices we pay and may vary at your institution
20 g L-ascorbic acid 2-phosphate
0.5 g insulin
0.5 g transferrin
2 mg sodium selenite
Sigma S5261-10G ($27.10/10 g)
4 mg FGF2-G3
Made by rPPC, $1290/80 mg for academic users, includes 1L culture of E. coli ($380), processing ($365), His purification ($380), SDS-PAGE analysis ($165)
10 µg NRG1
10 µg TGFB3
Total for 100 L of B8
Including a 1-liter bottle of DMEM/F12 (Corning 10-092-CM, $6.74), in-house B8 costs $10.02 per liter.
Other reagents and consumables
Autoclaved Milli-Q water, we use autoclavable Nalgene polycarbonate bottles (Thermo, 362015-0500)
50 mL Falcon conical centrifuge tube (Corning, 352098 = $0.12)
Sodium hydroxide (NaOH) (1 N), Sigma S2770 = $14.85)
Hydrochloric acid (HCl) (1 N), Sigma H9892 = $19.35)
250 mL PES 0.2 µm bottle top filters (Corning 431096 = $3.28 each)
2 mL microfuge tubes, sterile (Axygen MCT-200-C-S = $23.93 pack of 250)
Sodium selenite (Sigma, S5261-10G), make a 2 mg/mL stock solution, (100 mg/50 mL sterile Milli-Q water), make 1 mL aliquots and store at -20 °C
Recombinant human FGF2-G3 (made by core), comes at ~5 mg/mL, store at -20 °C
Recombinant human NRG1 (Peprotech 100-03-250UG), dilute to 100 µg/mL in water, store at -20 °C
Recombinant human TGFB3 (Cell Guidance Systems GFH109-100), comes as a liquid stock at 250 µg/mL, store at 4 °C
If you have any suggestions, know cheaper suppliers, have ideas to make the preparation simpler, or have any comments please contact us.