B8 consists of a 1-liter bottle of DMEM/F12 (Corning) and a 1.5 mL supplement aliquot.
We standardly used the 'low-cost, easier to make' variant of B8 from Fig. S5C (Kuo et al., 2020).
It should take less than 1 hour to make 100 aliquots of supplement.
Using FGF-G3 prepared by the Northwestern Protein Production Core it is possible to make B8 supplement in a single tube.
Single supplement aliquot
Place 47 mL of Cell Culture Water into a 50 mL Falcon tube. Add 20 g of L-ascorbic acid 2-phosphate. Mix until dissolved.
Place 48 mL of Cell Culture Water into a 50 mL Falcon tube. Add 0.5 g of insulin, mix, add 400 µL of HCl, mix until dissolved. Add 525 µL of NaOH (to pH 7.1), mix until dissolved.
Place 41 mL of Cell Culture Water into a 50 mL Falcon tube. Add 0.5 g of Optiferrin, 1 mL of 2 mg/mL sodium selenite, 7 mL of 0.6 mg/mL FGF2, 40 µL of 250 µg/mL TGFB3, and 100 µL of 100 µg mL NRG1, mix until dissolved.
Add all three tubes to a single bottle-top filter and sterilize, make 1.5 mL aliquots. Freeze at -20 °C
Costs to make supplement for 100 liters
Prices updated Jan 2020
All prices are based on list price but do not include shipping. These prices will likely be less 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 ($32.10/10 g)
4 mg FGF2-G3
Made by rPPC (~$1688/80 mg) prices for external academic users, includes 1L culture, His purification, SDS-PAGE analysis
10 µg NRG1
10 µg TGFB3
Total for 100 L of B8
Including a 1 liter bottle of DMEM/F12 (Corning 10-092-CM, $10.38), in-house B8 costs $13.92 per liter.
The cost of DMEM/F12 seems to vary between vendors. List prices are significantly higher (Corning = $181 per case of 6, ~$30 per liter).
Other reagents and consumables
Cell Culture Water, room temperature (500 ml, Corning 25-055-CV = $6.63)
50 mL Falcon conical centrifuge tube (Corning, 352098 = $0.19)
Sodium hydroxide (NaOH) (1 N), Sigma S2770 = $14.85)
Hydrochloric acid (HCl) (1 N), Sigma H9892 = $19.35)
150 mL PES 0.2 µm bottle top filters (Corning 431153 = $4.75)
1.5 mL microfuge tubes, sterile (Corning Axygen MCT-150-C-S = $12.29)
Sodium selenite (Sigma, S5261-10G), make a 2 mg/mL stock solution, (100 mg/50 mL Cell Culture Water), make 1 mL aliquots and store at -20 °C
Recombinant human FGF2-G3 (made by core), dilute to 4 mg/mL, store at -20 °C
Recombinant human NRG1 (Peprotech 100-03-250UG), dilute to 100 µg/mL, 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.