Comparison of thyroidectomized calf serum and stripped serum for the study of thyroid hormone action in human skin fibroblasts in vitro.
BACKGROUND
Knowledge on the action of thyroid hormone (TH) is augmented by the study of tissue responses to TH in vitro. In order to support the growth of cells in vitro, calf serum (CS) is usually added to the medium to provide necessary nutrients and growth factors. However, the content of endogenous TH in the CS may obfuscate changes with small doses of TH. We therefore compared the use of TH-depleted medium, either by resin treatment (stripped-CS) or by the use of CS from a thyroidectomized calf (TxCS) for gene expression studies.


METHODS
We describe the method for preparing a thyroidectomized calf, harvesting the blood and preparing the serum. We utilized methimazole in conjunction with the thyroidectomy to prevent TH synthesis in the event of regrowth of the thyroid remnant.


RESULTS
Total triiodothyronine (T(3)) and thyroxine concentrations in TxCS were low at <30 ng/dL and <1 microg/dL, respectively. We compared the effect of T(3) on basic transcription element-binding protein (BTEB)1 and stanniocalcin (STC)-1 mRNA expression in human fibroblasts from a normal individual and a subject with resistance to TH (RTH) cultured in stripped-CS to TxCS and demonstrated that with stripped-CS and TxCS differences in the BTEB1 and STC-1 expression of normal and RTH fibroblasts could be detected.


CONCLUSIONS
Both stripped-CS and TxCS are suitable to detect subtle differences in TH responsiveness between normal and RTH human skin fibroblasts, yet TxCS is not as costly as stripped-CS and relatively easy to prepare.
Moeller
Lars C
LC
Department of Medicine, University of Chicago, Chicago, Illinois 60645, USA.
Wardrip
Craig
C
Niekrasz
Marek
M
Refetoff
Samuel
S
Weiss
Roy E
RE
D000818:Animals; D013956:Antithyroid Agents / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects*; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008713:Methimazole / Q000494:pharmacology; D018962:Phlebotomy; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044967:Serum / Q000737:chemistry*; D012867:Skin / Q000166:cytology* / Q000187:drug effects; D013963:Thyroid Hormones / Q000097:blood / Q000494:pharmacology*; D013965:Thyroidectomy
D013956:Antithyroid Agents; D003470:Culture Media; D004273:DNA, Neoplasm; D006023:Glycoproteins; C489676:KLF9 protein, human; D051741:Kruppel-Like Transcription Factors; D012334:RNA, Neoplasm; D013963:Thyroid Hormones; D008713:Methimazole; C051795:teleocalcin