how to charcoal strip media - need to remove hormones (Apr/30/2009 )

Hi All,

Does anyboidy have a pretty straight forward protocol for stripping media (supp with serum) of hormones

Most papers I have read just say media was charcoal stripped, but i dont know how to do this.

We are looking into the effects of oestrogens and also man-made oestrogens (a group of chemicals called EDCs) on our cells so we need to remove all sources of these chemicals from our media before testing

I dont think the total removal of FCS is an option, as we do need some of the growth factors etc it provides

So can anybody provide me with an easy protocol for charcoal stripping media for removal of hormones or even a protocl for removal of other chemicals that may work.

Thanks,

Cotchy.

cotchy on Apr 30 2009, 10:22 AM said:

I usually used dextran-coated charcoal.

From one of my M & Ms: "Dextran-coated charcoal was prepared by suspending 0.5 g activated carbon (Norit A, Norit Americas Inc, Marshall, TX) in 20 mL of 2.5 mg/mL Dextran T40 (GE Healthcare, Oslo, Norway) and incubating the suspension at 56 oC for 30 minutes. The charcoal was recovered by centrifugation at 5,000 x g for 10 minutes and washed twice with 50 mL water. FCS (25 mL) was then added to the pellet and the suspension adjusted to pH 3.0 with 5 mol/L HCl. Following incubation at 56 oC for 30 minutes with occasional agitation, the charcoal was removed by centrifugation (20,000 x g, 20 mins, 20 oC) . After titration to pH 6.8 with 5 mol/L NaOH, the solution was filter sterilized".

Hope this helps.

Wow its pretty complicated, i hoped it would be way easier, but if thats the way to do it then thats the way to do it.

thanks for your help.

Cotchy.

cotchy on May 1 2009, 09:57 AM said:

A more straightforward procedure is that described by Chen.
I have not tried it myself but it may be adequate for your purposes.

Chen RF Removal of fatty acids from serum albumin.... (1967) J. Biol Chem 242: 173-81.

If your lab has the money, you can always buy commercially manufactured "charcoal stripped FBS". Saves the work, but at $80 for 50 ml (Invitrogen) rather pricey!

As you are probably aware, it may be wise to use media without phenol red. This indicator is reported to be contaminated with low levels of a lipophilic estrogen mimic.

Hope this helps.

Yep many thanks Klinmed, i was aware that phenol red acts as an oestrogen mimic se we use phenol red free media, thanks for the reference to the paper.

There should be other sources of stripped serum. Manufacturers of diagnostics used serum for creation of their test kit calibrators.