Hi, i have to treat my MCF7 cells with tamoxifen, to get ER+ tamoxifen resistant cells, i need to use steroid depleted FBS, it is expensive, so can any body advice about how to prepare it from usually used FBS? Thanx
You need to buy activated charcoal- Sigma Chem Co. used to sell it. You can autoclave a funnel and whatman I paper. Fill it with activated charcoal- I no longer remember the ratio of charcoal to FBS. You can purchase heat inactivated FBS and filter this through the activated charcoal under aseptic conditions. Filter sterilize FBS using a 0.2 micron filter. This was good enough for steroid hormones. The commercial variety you can buy is charcoal and dextran stripped FBS. Hope this helps...
To make charcoal or hormone-stripped or -depleted fetal bovine serum (FBS)...
To heat inactivated fetal bovine serum, add activateded charcoal to a final concentration of 1% (Sigma #C9157) and Dextran T-70 (MW ~70KDa; Sigma#D4751 ) to a final concentration of 0.1% (5 g and 0.5g respectively/500 ml bottle of serum)
Incubate mixture for 1 hr at room temperature with agitation (using a platform shaker at 100 rpm, or similar)
Pellet charcoal by centrifuging at 12,000xg for 15 min.
Vacuum filter supernatant through at Whatman #4 filter (110 mm).
Filter sterilize filtered supernatant with a 0.2 µM sterile filter.
Aliquot and store at -20°C prior to use
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