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, 100 µL of 100 µg/mL TGFB1, 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
NOTE: Peprotech has not maintained discounts previously provided over the last decade therefore we have sought alternative suppliers. Either TGFB1 or TGFB3 can be used, we are currently using TGFB1 as it is cheaper.
Costs to make enough supplement for 100 liters
Prices updated Aug 2021
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 ($28.25/10 g)
4 mg FGF2-G3
Made by rPPC, $1280/100 mg for academic users, includes 1L culture of E. coli ($390), processing ($380), His purification ($340), SDS-PAGE analysis ($170)
10 µg NRG1
10 µg TGFB1
Qkine Qk010_TGFB1_100µg ($620/100 µg)
Total for 100 L of B8
At current prices, including a 1-liter bottle of DMEM/F12 (Corning 10-092-CM, $7.02), 1 liter of B8 costs $10.40 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), dilute to 100 µg/mL in water, store at -20 °C
Recombinant human TGFB1 (Qkine), dilute to 100 µg/mL in 10 mM HCl, store at -20 °C
If you have any suggestions, know cheaper suppliers, have ideas to make the preparation simpler or have any comments please contact us.