Hello all,
I am working with hormone responsive cancer cells (MCF7, ER positive) and wondering whether I need to use stripped serum or not .
In literature, some people either strip their serum to deplete it from undefined levels of hormones like estrogen and then add it back but sometimes they do not stripp serum indicating that fetal serum does not contain hormones in contrast to bovine serum .
Does anyone have ever done some analysis or could tell me where this has been demonstrated or what I could refer to once I have to prove it?!
Thanks for any suggestion
Generally, fetal bovine serum (FBS) is rich in a diverse range of hormones/ growth factors. This quality is desirable and explains why demand and cost is so high for FBS. Adult animal serum will generally contain fewer of the desirable growth factors, but will contain hormones. Serum contents are highly variable in all cases. If you wish to know the contents of your serum you can either test it yourself, or buy pre-tested serum from a reliable vendor. If you contact your vendor, and give them the lot number of your serum, they will be generally be able to provide this information for you. It would probably be of little use to have the test results from someone else's serum.
Amended on 17 October 2017
Since usual hormone assays based upon affinity of immunoglobulins (ELISA) are not quantitative at all, study using hormone should be performed by using reliable HPLC method. CLIA (Chemiluminescent Immunoassay) method and other immunoassays are also sadly making many erroneous results. About 2,000-fold difference has been occurred between HPLC method and notorious ELISA method (please see files; JCB fucoidan transport, Tokita ELISA, and ELISA fucoidan). Then, estradiol-17β concentration in female serum measured by immunological method (160 pg/mL) may become 0.32 μg/mL. The reliable HPLC method for hormone assay has also been mentioned recently (please see file; The Fascio Effect). As an example, serum free biotin concentration, which has not yet been considered as hormone but has a similar growth factor (vitamin H) as hormone, has been determined as 0.122 μg/mL by using reliable HPLC method (please see file; Wide range of Biotin).
Therefore, I describe the reliable results using HPLC-determination methods.
I have searched five liver tissues by using the new quantitative proteomics of the PDMD (Protein-Direct-Microsequencing-Deciphering) method. Only three specimens have steroid-hormone related proteins among 21 specimens. HCC tissue with PBC (the aged woman) has Androgen receptor at 7.1 μg/mg of tissue protein and Steroid hormone receptor ERR2 at 2.2 μg/mg of tissue protein. LC tissue with leprosy (the aged woman) has Mineralocorticoid receptor at 0.19 μg/mg of tissue protein. Healed HepG2 cells by fucoidan (15y male) have Glucocorticoid receptor/GR at 2.2 μg/mg of cell protein (please see file; HepG2 fucoidan).
In order to clarify the age dependent changes in expression of peptide-hormone related proteins, I have searched fetal hepatocyte Hc cells by using PDMD method.
Only two peptide hormone receptors have been found; i.e.,
Relaxin receptor 2 at 9.0 μg/mg of cell protein and Melanocortin receptor 5/MC5-R at 4.1 μg/mg of cell protein. Total hormonal proteins becomes 13.1 μg/mg of cell protein.
On the other hand, many differentiation or developmental proteins (hormones?) have been found.
Cadherin EGF LAG seven-pass G-type receptor 3/Flamingo homolog 1/EGF-like protein 1/Cadherin family member 11 at 0.87, Plancental-specific 1-like protein/Placenta-specific protein 1 at 24.4, Desmoyokin/Neuroblast differentiation-associated protein AHNAK at 19.9, Actinin-associated LIM protein/PDZ and LIM domain protein 3/Alpha-actinin-2-associated LIM protein at 28.5, Fetal-inflammatory-biotinidase at 0.2, Coiled-coil and C2 domain-containing protein 1B/Freud-2 at 2.7, Delta-like protein 4/Drosophila Delta homolog 4 at 3.0, Dyslexia susceptibility 1 candidate gene 1 protein/Dynein assembly factor 4, axonemal at 16.2, Early activation antigen CD69/Activation inducer molecule at 7.8, Interferon-induced protein with tetratricopeptide repeats 3/Retinoic acid-induced gene G protein at 3.0, Jumonji domain-containing protein 2B/Lysine-specific demethylase 4B at 8.9, Notch homolog 2 N-terminal-like protein at 13.4, Protein kinase C eta type at 40.2, Protein SCD6 homolog/Protein FAM61A/Protein LSM14 homolog A at 20.1, SLIT and NTRK-like protein 1/Leucine-rich repeat-containing protein 12 at 1.2, Transformation-related gene 15 protein at 1.4, Tripartite motif-containing protein 42 at 2.0 μg/mg of cell protein (total 19.2% of cell protein).
We have further compared hormone concentrations and developmental proteins in human serum between child (4mo-4y) and adult (22y-52y).
Hormones and hormone receptors have been detected as follows; i.e.,
4mo boy has Antisense basic fibroblast growth factor B/Nucleoside diphosphate-linked moiety X motif 6 at 6.4 μg/mg of serum protein and Neurosecretory protein VGF at 1.5 μg/mg of serum protein (total 7.9 μg/mg of serum protein).
7mo boy has Antisense basic fibroblast growth factor B/Nucleoside diphosphate-linked moiety X motif 6 at 6.4 μg/mg of serum protein (total 6.4 μg/mg of serum protein).
8mo girl has Fibroblast growth factor 13/Fibroblast growth factor homologous factor 2 at 4.0 μg/mg of serum protein (total 4.0 μg/mg of serum protein).
1y girl A has Insulin-like growth factor 1A precursor/IGF-I at 6.7 μg/mg of serum protein (total 6.7 μg/mg of serum protein).
1y girl B has no hormone in her serum.
1y4mo girl B has Growth/differentiation factor 8/GDF8/Myostatin at 11.9 μg/mg of serum protein and Activin receptor type-2B at 2.6 μg/mg of serum protein (total 14.5 μg/mg of serum protein).
1y11mo girl B has Atrial natriuretic peptide receptor A at 2.0, C-C motif chemokine 17/CCL17/CC chemokine TARC/Small-inducible cytokine A17/Thymus and activation-regulated chemokine at 6.2, and Pro-epidermal growth factor/Urogastrone at 1.2 μg/mg of serum protein, respectively (total 9.4 μg/mg of serum protein).
3y4mo girl has Interleukin-9 receptor at 4.5 μg/mg of serum protein (total 4.5 μg/mg of serum protein).
4y girl has Epidermal growth factor receptor/EGF receptor at 3.6 μg/mg of serum protein and Tyrosine-protein kinase receptor ETK1/Ephrin type-A receptor 3 at 1.6 μg/mg of serum protein (total 5.2 μg/mg of serum protein).
On the other hand, adolts’ serum has as follows; i.e.,
22y female has Cartilage-derived morphogenetic protein 1/Growth/differentiation factor 5/Bone morphogenetic protein 14 at 8.6 μg/mg of serum protein (total 8.6 μg/mg of serum protein).
32y female has Epidermal growth factor receptor 5/EGFR-5 at 1.5 μg/mg of serum protein and Epidermal growth factor-like 2/Flamingo homolog 3 at 5.9 μg/mg of serum protein (total 7.4 μg/mg of serum protein).
33y male has Activin receptor type IIA at 8.3 μg/mg of serum protein (total 8.3 μg/mg of serum protein).
52y female has Interleukin-31 receptor subunit alpha at 3.5 μg/mg of serum protein (total 3.5 μg/mg of serum protein).
The median difference in hormone-related proteins is not present between child and adult (n1 = 4, n2 = 9; Mann-Whitney’s U test).
On the other hand, content of developmental proteins are also compare; i.e.,
4mo boy has developmental proteins at 3.3 % in his serum.
7mo boy has them at 1.8 %.
8mo girl has them at 9.0%.
1y girl A has them at 6.5 %.
1y girl B has them at 9.2 %.
1y4mo girl B has them at 5.1 %.
1y11mo girl B has them at 3.4 %.
3y4mo girl has them at 5.8 %.
4y girl has them at 17.7 %, but she has been transplanted her liver from her father liver at age 3y10mo. It is interesting that quickly growing fetal hepatocyte Hc has 16.8% of Development proteins similar to serum of patient with regenerating quickly-growing liver.
Adults’ serum has them as follows; i.e.,
22y female has at 3.1 %.
32y female has at 2.2 %.
33y male has at 1.8 %.
52y female has at 3.2 %.
The median differences is present in development proteins between child and adult (p < 0.05; n1 = 4, n2 = 8; median of adult is 2.7 % and median of child is 5.5 %; Mann-Whitney’s U test, two-tailed test). Therefore, developmental proteins in children's tissue and serum seem to be higher than adult. But, hormone differences may not be present after the parturition or birth.
Deveolpmental proteins found in serum are as follows; i.e.,
4mo boy has Janus kinase and microtubule-interacting protein 1/GABA-B receptor-binding protein at 3.1, Geminin at 3.3, Patched domain-containing protein 3/Patched-related protein at 5.1, Piwi-like protein 4 at 13.0, Protein bicaudal C homolog 1 at 5.4, and Tetratricopeptide repeat protein 17/TPR repeat protein 17 at 2.8 μg/mg of serum protein, respectively.
7mo boy has Abnormal spindle-like microcephaly-associated protein homolog/Abnormal spindle protein homolog at 6.6, Dedicator of cytokinesis protein 3/Modifier of cell adhesion/Presenilin-binding protein at 3.9, Neuronal acetylcholine receptor subunit alpha-10 at 2.5, Uncharacterized protein C12orf64/Otogelin-like protein at 5.2 μg/mg of serum protein, respectively.
8mo girl has PH (Pleckstrin homology)-interacting protein/IRS-1 PH domain-binding protein at 17.5, TCEA-like protein 5/Transcription elongation factor A protein-like 5 at 6.1, Testis development protein NYD-SP11/WD repeat-containing protein 87 at 5.8, Testis development protein NYD-SP11 at 5.1, Structural maintenance of chromosomes flexible hinge domain-containing protein 1 at 12.7, TCEA-like protein 5/Transcription elongation factor A protein-like 5 at 6.1, Rabconnectin-3 beta/WD repeat-containing protein 7 at 6.6, Probable helicase with zinc finger domain at 3.8, Nephrin/Renal glomerulus-specific cell adhesion receptor at 3.6, Laryngeal carcinoma-related protein 1/Gametogenetin-binding protein 2 at 8.6, Kinesin-like protein KIF 27 at 4.5, Intestine and liver tetraspan membrane protein/Transmembrane 4 L6 family member 4 at 2.3, Forkhead box protein D3/HNF3/FH transcription factor genesis at 7.0, Exchange factor for ARF6/Pleckstrin homology and SEC7 domain-containing protein 1 at 34.6, Atrophin-1-like protein/Arginine-glutamic acid dipeptide repeats protein at 9.4 μg/mg of serum protein, respectively.
1y girl A has Agrin at 16.7, Gastric cancer antigen Ga19/N-alpha-acetyltransferase 15 at 13.0, Helicard/Interferon-induced helicase C domain-containing protein 1 at 2.5, Peroxisome proliferator-activated receptor alpha at 13.5, Rabenosyn-5/RAB effector RBSN at 5.8, Recessive polycystic kidney disease protein Tg737 homolog at 3.7, Testis development protein NYD-SP9/Microfibrillar-associated protein 3-like at 10.1 μg/mg of serum protein, respectively.
1y girl B has A-kinase anchor protein 13/Breast cancer nuclear receptor-binding auxiliary protein at 6.2, Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform/PI3-kinase p110 subunit beta at 4.2, Williams-Beuren syndrome chromosomal region 11 protein/General transcription factor III at 12.6, Tetraspanin-32/Tspan-32/Protein Phemx at 10.5, Tenascin-X at 10.8, RING finger protein unkempt homolog /Zinc finger CCCH domain-containing protein 5 at 3.4, Serine protease HTRA4 at 6.9, Jerky protein homolog-like at 1.6, Guanine nucleotide exchange factor DBS at 19.4, Down syndrome cell adhesion molecule at 9.8, A-kinase anchor protein 13/Breast cancer nuclear receptor-binding auxiliary protein at 6.2 μg/mg of serum protein, respectively.
1y4mo girl B has SH2B adaptor protein 2 at 2.6, C-1-Tetrahydrofolate synthase, cytoplasmic/C1-THF synthase at 8.0, Low-density lipoprotein receptor-related protein 1B/LRP-1B at 7.7, Cysteine-rich motor neuron 1 protein at 10.6, Collagen alpha-1(XVII) chain/120 kDa Linear Ig A disease antigen at 12.8, Abl interactor 2 at 9.5 μg/mg of serum protein, respectively.
1y11mo girl B has Angio-associated migratory cell protein at 7.7, Insulin-responsive sequence DNA-binding protein 1/IRE-BP1/Sushi, nidogen and EGF-like domain-containing protein 1 at 2.4, Latent-transforming growth factor beta-binding protein 2 at 9.6, Protein furry homolog at 8.3, Ras-interacting protein 1 at 4.0, and Sperm-specific protein/C-Jun-amino-terminal kinase-interacting protein 4 at 2.3 μg/mg of serum protein, respectively.
3y4mo girl has Retinoic acid-induced protein 2 at 4.4, G Patch domain-containing protein 1/Evolutionarily conserved G-patch domain-containing protein at 2.7, Leucine-rich repeat transmembrane neuronal protein 1 at 11.7, Immunogloblin superfamily member 3/Glu-Trp-Ile EWI motif-containing protein 3 at 3.7, Histone-lysine N-methyltransferase MLL3/Lysine N-methyltransferase 2C at 9.3, HBxAg transactivated protein 2/Protein PRRC2C/HBV X-transactivated gene 2 protein at 8.6, HBV X-associated protein 1/AH receptor-interacting protein at 1.7, Embryonal Fyn-associated substrate at 7.0, Dyslexia susceptibility 1 candidate gene 1 protein at 3.0, and Contactin-5 at 5.7 μg/mg of serum protein, respectively.
4y girl has Protein NDRG1/N-myc downstream-regulated gene1 protein/Differentiation-related gene 1 protein at 4.7, Membrane-spanning 4-domains subfamily A member 10 at 42.0, Paternally-expressed gene 3 protein at 4.0, Protein FAM122B at 12.6, Renal carcinoma antigen NY-REN-36/Zinc finger protein 608 at 4.1, Signal-induced proliferation-associated protein 1 at 18.3, Teneurin-3/Tenascin-M3 at 7.8, and von Willebrand factor C domain-containing protein 2-like at 83.0 μg/mg of serum protein, respectively.
Adolts’ serum has them as follows; i.e.,
22y female has Nuclear receptor subfamily 6 group A member 1/Germ cell nuclear factor at 4.6, Olfactomedin-1/Neuronal olfactomedin-related ER localized protein/Noelin at 3.3, Prominin-1 at 4.4, and Tudor domain-containing 4, isoform CRA_b at 9.5 μg/mg of serum protein, respectively.
32y female has Lectomedin-3/Latrophilin-3/Adhesion G protein-coupled receptor L3 at 3.7, Sodium channel protein type 8 subunit alpha at 5.3, T-complex protein 10A homolog at 11.9, Testis development protein NYD-SP11/WD repeat-containing protein 87 at 7.0, and Transmembrane protein 65 at 3.3 μg/mg of serum protein, respectively.
33y male has B-cell linker protein at 2.2, Protein Wnt5a at 9.1, and Zinc finger protein 185 at 6.9 μg/mg of serum protein, respectively.
52y female has Chondroitin sulfate proteoglycan 5 at 2.7, F-box only protein 38 at 10.9, Neurogenic locus notch homolog protein 3 at 5.5, Ovarian cancer-related protein 10-2/Teashirt homolog 2 at 3.1, Semaphorin-6A at 5.5, Uromodulin-like 1/Olfactorin/UMODL1 at 2.0, and Zinc finger homeobox protein 4 at 5.0 μg/mg of serum protein, respectively.
Hello Vanessa,
Actually, FBS does contain low levels of estrogens. You also need to know that phenol red has some estrogenic activity. I used to work regularly with MCF-7 cells before. We were using phenol red-containing medium with regular FBS for the maintenance of the cell line. When we were planning an experiment involving estrogen receptor signaling, we were pre-starving a subculture in phenol red-free medium containing charcoal-dextran stripped FBS for one passage before seeding the cells for the experiment in the same estrogen-free medium. That ensures that you get the maximal sensitivity on estrogen induced signaling pathways detection when you compare your estrogen treated sample to your untreated control.
Regards.
Thanks Mr Hayakawa,
unfortunately your answer is not really helpful for me as someone who is not familiar with the method, thanks anyway.
@Martine Bail :
So I use white Medium and informed about stripping serum, but wondered if I could avoid it ....
I let the cells that were maintained in red meidum with normal FBS now 48h in white Medium with stripped serum.
Their cytoplasm enlarged pretty much and they look unhealty...... did you also notice that ?
Since MCF-7 are dependent on estrogens to proliferate, you expect them to drastically reduce their growth in estrogen-free medium. That's a sign that your starving is actually working. I suppose that the morphological changes that you see are probably due to their quiescence.
Of note : we were also using phenol-red free trypsin-EDTA for the passaging. That's particularly important for the pre-starved subculture.
Amended on 17 October 2017
Normal fetal hepatocyte Hc has hormonal proteins at 1.3% and developmental proteins at 19.2% of cell proteins.
It has been also found that Hepatoma HepG2 (without fucoidan; derived from HCC tissue of 15y Caucasian male) has hormonal proteins at 1.9% and developmental proteins at 7.1% of cell proteins. Healed HepG2 (with fucoidan at 0.102 mg/mL for three days) has hormonal proteins at 1.6% and developmental proteins at 9.7% of cell proteins.
It seems that hormone-related proteins seem to be similar between fetus liver and adult liver, but development-related proteins seem to be higher in the fetal liver cells than adult liver cells. Therefore, the use of FCS instead of adult bovine serum in cell-culture medium is not neccessary. Adult serum has much connectin/titin (usually Mr larger than 800,000), and this adult serum may be heat treated, filtered, and used instead of using the blood of miserable fetal bovine (please see file; JMBT alopecia). Further, the healing effect of Fucoidan seems to be not related to the gene expression of hormone-related protein and developmental proteins as previously declared in our report (please see file; HepG2 fucoidan).
Amended on 20 October 2017
Furthermore, it has been found that steroid hormones (estrogen etc.), alkaloides (caffeine, nicotine, capsaicin etc.), and alcohol (ethanol) are not linked to the cancer at all, but some peptide hormones such as insulin (INS), insulin-like growth factor I (IGF-I), prolactin (PRL), and growth hormone (GH) seem to be strongly linked to the cancer as estimated by our PDMD method. It is noteworthy that these receptors all have protein tyrosine kinase (PYK) activity.
HCC liver with PBC (female) has Insulin-like growth factor-binding protein 5/IGFBP-5 at 3.0 μg/mg of tissue protein and Insulin-like growth factor binding-protein 3/IGFBP-3 at 0.7 μg/mg of tissue protein, and Growth hormone receptor/GH receptor at 12.4 μg/mg of tissue protein. Hepatoma HepG2 (without fucoidan; male) has Insulin receptor substrate 2/IRS-2 at 15.0 μg/mg of cell protein. HCC liver (named No.6; male) has Prolactin receptor type 2 at 6.4 μg/mg of tissue protein and Insulin-degrading enzyme/Insulinase/IDE at 13.3 μg/mg of tissue protein.
Healed HepG2 (normal) has IGF-II receptor/CI Man-6-P receptor at 11.3 μg/mg of cell protein. IGF-II receptor is GPCR, which seems safe to be not related to cancer.
Heavy alcohol drinker of mammal of Scandentia Tupai (Treeshrew) seems to be healthy without any cancer.
Liver of pseudo cancer (non cancer tissue) has Cannabinoid receptor 1/CB1/CB-R (GPCR) which can also bind to nicotine, but other cancer tissues have it not. Fetal hepatocyte Hc and hepatoma HepG2（grown without fucoidan) has no Nicotinic-AChR (cation-transport related), but healed HepG2（with fucoidan) has Nicotinic-acetylcholine receptor protein subunit delta. LC tissue with leprosy has Nicotinic-acetylcholine receptor protein, delta chain, Neuronal acetylcholine receptor protein, alpha-5 chain, and Neuronal acetylcholine receptor protein, beta-4 chain. LC tissue (designated as No. 6) has Acetylcholine receptor protein, beta chain and Acetylcholine receptor protein, epsilon chain, but HCC tissue of this patient also has Neuronal acetylcholine receptor protein alpha-5 chain. Thus, Nicotine is not linked to the cancer at all.
Hepatoma HepG2 has Insulin receptor substrate 2/IRS-2 at 15.0 μg/mg of cell protein and GRB2-associated-binding protein 1/Growth factor receptor bound protein 2-associated protein 1 at 1.2 μg/mg of cell protein. Fucoidan derived from Japanese edible Mozuku has inhibited expression of these proteins (please see file again; HepG2 fucoidan). This may be the additive reason why Fucoidan has reduced the growth of HepG2 cells (growth rate has been reduced to 33% as compared to no addition control at 0.102 mg/mL for 3-day therapy). It has been firstly found that fucoidan reduces the other growth factor of biotin (vitamin H) as estimated biotinidase enzyme-kinetics (please see file; Anticancer Res), though biotinidase amount is not changed (please see file; HepG2 fucoidan).
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