FBS Charcoal Stripping Protocol: How to Prepare Hormone-Depleted Serum

Fetal bovine serum contains steroid and thyroid hormones, vitamins and other lipophilic molecules. For studies on oestrogen, androgen, glucocorticoid or thyroid hormone signalling, these background hormones must be removed, otherwise the effect of the hormone you add cannot be measured cleanly. The standard method is treatment with dextran-coated charcoal (DCC), known as charcoal stripping.

This protocol describes a widely used laboratory method. If you need larger volumes or consistent, documented lots, SeamlessBio offers single and double charcoal treatment as a serum processing service.

What charcoal stripping removes – and what it doesn’t

Activated charcoal adsorbs small, lipophilic molecules. The dextran coating partly covers the pores, so large proteins such as albumin, immunoglobulins and most growth factors stay in the serum.

Reduced by charcoal stripping: – steroid hormones (oestradiol, progesterone, testosterone, cortisol) – thyroid hormones (T3, T4) – lipophilic vitamins and retinoids – some lipids, fatty acids and lipophilic drug residues – some growth factors are reduced as well, to a lesser extent

Largely retained: – albumin, transferrin, immunoglobulins – most high-molecular-weight proteins

Charcoal stripping vs. delipidation: Delipidation removes lipids but leaves steroid hormones in the serum. If you need lipid-depleted serum with intact hormones, delipidation is the better choice. For human matrices see Human Serum Delipidated.

Materials

• FBS, e.g. FBS Standard or FBS Premium, thawed at 2–8 °C
• Activated charcoal, acid-washed, cell culture or analytical grade
• Dextran, average MW approx. 70 kDa (dextran T-70)
• Buffer for the DCC suspension, e.g. 10 mM HEPES or Tris-HCl pH 7.4 with 0.25 M sucrose and 1.5 mM MgCl₂
• Centrifuge and tubes or bottles suitable for the volume
• Water bath at 56 °C (only for the stringent variant)
• Filters: 0.45 µm prefilter and 0.2 µm sterile filter, low protein binding (PES)

Step-by-step protocol

1. Prepare the DCC suspension (day before)

1. Prepare a suspension of 0.25 % (w/v) activated charcoal and 0.025 % (w/v) dextran T-70 in buffer. Example: 2.5 g charcoal + 0.25 g dextran in 1 L buffer.
2. Stir overnight at 2–8 °C so the dextran coats the charcoal.

2. Pellet the charcoal

3. Transfer a volume of DCC suspension equal to the volume of serum you want to strip into a centrifuge tube or bottle.
4. Centrifuge at about 500 × g for 10 minutes. Carefully remove and discard the supernatant. The charcoal pellet stays in the tube.

3. First stripping round

5. Add the thawed serum to the charcoal pellet and resuspend completely by vortexing or shaking.
6. Incubate with gentle, continuous mixing. Choose one variant:
   • Gentle: 12–16 hours (overnight) at 2–8 °C
   • Stringent: 45 minutes at 56 °C. This also heat-inactivates the serum (see heat inactivation protocol).
7. Centrifuge at about 500–1,000 × g for 10–15 minutes and transfer the serum to a new tube that contains a fresh charcoal pellet (prepared as in step 2).

4. Second stripping round

8. Resuspend and incubate again using the same variant as in round 1. A second round lowers residual hormone levels considerably. “Double charcoal” serum is stripped twice.

5. Remove the charcoal

9. Centrifuge at a higher speed, e.g. 2,000–3,000 × g for 20 minutes, to remove the charcoal as completely as possible.
10. Prefilter the serum through 0.45 µm, then sterile-filter through 0.2 µm. Fine charcoal particles clog filters quickly, so plan for several prefilters.

6. Aliquot and store

11. Aliquot the stripped serum and store it at −20 °C. Avoid repeated freezing and thawing. Label as “charcoal stripped”, with date and lot.

7. Quality control

12. Measure the target hormones (e.g. oestradiol, testosterone, cortisol) before and after stripping, by immunoassay or LC-MS/MS. Each lab defines its own acceptable residual level.
13. Test growth: stripped serum supports cell growth less well than untreated serum. Compare growth and morphology of your cell line before using the serum in experiments.

Practical tips

• Use phenol red-free medium for oestrogen studies. Phenol red has weak oestrogenic activity and undoes part of the benefit of stripping.
• Thaw serum correctly: at 2–8 °C overnight, not at 37 °C or higher.
• Keep conditions constant within a study. Charcoal type, incubation time and number of rounds all affect the result.
• Consider serum-free systems: If your goal is a defined, hormone-free background, a serum-free medium can be an alternative to stripped serum.

FAQ

What is charcoal-stripped FBS used for? For experiments in which steroid or thyroid hormones in the serum would mask the effect of added hormones. Typical examples are oestrogen and androgen receptor assays, glucocorticoid studies, nuclear receptor reporter assays and the preparation of steroid-free calibrator matrices.

What is the difference between single and double charcoal treatment? Single treatment means one incubation with charcoal; double treatment means two consecutive incubations with fresh charcoal. Double treatment gives lower residual hormone levels.

Why is the charcoal coated with dextran? The dextran layer partly covers the pores of the charcoal. Small molecules such as steroids are still adsorbed, while large proteins such as albumin are retained in the serum.

Does charcoal stripping change the growth properties of the serum? Yes, in most cases cells grow somewhat more slowly, because hormones, vitamins and some growth factors are removed. Test your cell line before using a new stripped lot.

Should I heat-inactivate before or after charcoal stripping? If you use the stringent variant at 56 °C, the serum is heat-inactivated during stripping. With the gentle 2–8 °C variant, heat-inactivate separately if your assay requires it.

Can SeamlessBio charcoal-strip serum for me? Yes. Single and double charcoal treatment for FBS and other sera are part of our serum processing service, from 100 mL. Prices and lead times on request – request a quote.

Related: Serum Processing Services · Fetal Bovine Serum · Human Serum Delipidated · FBS Dialysis Protocol · Serum-Free Systems