Protocol for Charcoal-stripping FBS to Deplete Hormones

To study the effects of steroid hormones in vitro without the confounding effects of hormones endogenous to the FBS
(or other sera), growth factors and cytokines may be depleted or effectively removed by charcoal stripping of serum. Incubation with charcoal, or charcoal-stripping, has been used to reduce estrogen levels in fetal bovine serum (FBS).
Dextran-coated charcoal is preferable for this method, because there is less unintentional loss of non-steroid serum
proteins compared to using uncoated charcoal.

Depletion of estrogen from fetal bovine serum (FBS) using the dextran-coated charcoal method, with and without and heat-inactivation

The following procedure will reduce the level of estrogen in FBS to < 10^-11 M , which corresponds to < 10^-12 M in the medium containing 10% FBS. Although this level is sufficiently low to permit most studies on estrogen-induced responses, it should not be assumed that such procedures completely eliminate either the estrogen or any other steroid from the serum or, indeed, the content of any other steroid family. It should be noted that heat inactivation of serum also removes factors other than the steroid. For example, in our studies of primary cultures, it was clear that, although the attachment of rat uterine cells to substrate was enhanced in FBS stripped with dextran-coated charcoal (DCC) at 4 °C, attachment was reduced threefold after DCC stripping at 56 °C. We have not characterized this attachment factor(s) but have found that it is consistently removed during heat inactivation of serum.
 

Preparation of charcoal-stripped serum

1. Incubate overnight at 4 °C Dextran-coated charcoal or Activated Charcoal Norit (final concentration 0.25%) and Dextran T-70 (final concentration 0.0025%) in 0.25 M sucrose/1.5 mM MgCl₂/10 mM HEPES, pH 7.4.
2. Use a volume of the dextran-coated charcoal (DCC) from step 1 equivalent to that of the serum which is to be stripped. Centrifuge it (500 x g for 10 min) to pellet the charcoal.
3. Decant the supernatant and replace it with the same volume of FBS. Remember that different lots of FBS, (even those from the same supplier), may have different growth characteristics and must be validated using your lab’s tissue culture protocol.
4. Vortex the tube to thoroughly mix the charcoal with the serum and incubate either:
   • for 12 h at 4 °C (DCC-stripped serum) or
   • for 2 x 45 min at 56 °C (heat-inactivated, DCC-stripped serum = HIDCC serum).*

   * Heat - inactivation is also used to prepare complement-deficient serum.

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