Northwestern University Feinberg School of Medicine

Paul Burridge Lab

Making B8 Supplement Aliquots

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

It should take less than 1 hour to make 100 aliquots of supplement, including aliquoting time.

NOTE: The Northwestern Recombinant Protein Production Core has closed down. We recommend making FGF2 in-house. 

Single supplement aliquot

Place 35 mL of sterile ultrapure 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 ultrapure 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 ultrapure 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 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 September 2022.

All prices are the actual prices we pay and may vary at your institution

20 g L-ascorbic acid 2-phosphate

Wako Chemicals USA 321-44823 ($165/100 g)

$33.00

0.5 g insulin

Gibco A11382ij ($900/5 g from Fisher Scientific

$90.00

0.5 g transferrin

InVitria Optiferrin 777TRF029-10G  ($1925/10 g) (or $1998 from Fisher Scientific)

$96.25

2 mg sodium selenite

Sigma S5261-10G ($36.70/10 g)

$0.01

4 mg FGF2-G3

Made in-house (~$120/200 mg)

$6.00

10 µg NRG1

Shenodoah 100-46-500ug ($769/500 µg)

$15.40

10 µg TGFB3

Qkine Qk054-0100 ($620/100 µg)

$62.00

Total for 100 L of B8

$302.66

At current prices, including a 1-liter bottle of DMEM/F12 (Corning 10-092-CM, $11.12), 1 liter of B8 costs $14.14 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)

 

Stock solutions

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, comes at ~5 mg/mL, store at -20 °C

Recombinant human NRG1, dilute to 100 µg/mL in water, store at -20 °C

Recombinant human TGFB1, dilute to 100 µg/mL in 10 mM HCl, store at -20 °C

 

Help

If you have any suggestions, know cheaper suppliers, have ideas to make the preparation simpler or have any comments please contact us