Planning to run my assay this week but couldn't figure out positive control for MTT assay. I want to use cells without treatment as negative control while needing an agent or growth factor that induces endothelial cell proliferation as positive control. Please your advise on this will be appreciated. Thanks
VEGF is a potent stimulator of endothelial cell proliferation and is used in many assays as a positive control. If you want to look at inhibition, Sunitinib is a receptor tyrosine kinase inhibitor that works well in cell assays. Other options include Cyclosporine A, Itraconazole and Orlastat.
VEGF (5-20ng/ml) will be a positive control for your MTT assay. However, if you want to pursue the cell cycle, BrdU assay is the standard tech. For the negative control, you can use a starved conditioned medium, such as M199+0.1% BSA. And treatment reagents can be diluted with this media.
I don't think that MTT assay is really a good instrument to measure cell proliferation, to my opinion the best way would be to measure the number of cells or cell nuclei or the DNA content of your culture.
I think you can use any confirmed agent that previously had shown cytotoxic effects on this cell line. Clearly, having the lowest IC50 is the best choice, as a comparison.
I agree with Dominique Duval: MTT is NOT a proliferation assay, but a NADH-dependent assay reflecting the cells' metabolism. There is not necessarily a correlation between OD-values and cell number - you may get different IC50 values. Also, untreated cells are not a "negative" control.
FGF-2 (5- 10 ng/ml) is another good positive control for endothelial cell proliferation or 10% FBS medium vs. serum free or low serum (0.5% or less) control. I favor BrdU ELISA as method to measure cell proliferation.
vascular endothelial growth factor (VEGF) is a good positive control. But I agree that MTT assay is not optimal for measuring cell proliferation. Better suited would be CellTiter 96® AQueousOne Solution Cell Proliferation Assay (Promega Corp., Madison, WI). Briefly, the phenazine ethosulfate (PES)-coupled reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to a purple, water-soluble formazan by living, undamaged cells is measured with this assay.
There are several options for positive controls that can be used for endothelial cell proliferation assays, such as the MTT assay. Here are a few suggestions:
It's important to note that the choice of positive control may depend on the specific experimental conditions and the cell type being used. Additionally, it's important to include appropriate negative controls and experimental replicates to ensure the accuracy and reproducibility of the results.
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Olayinka Olajiga
A positive control for endothelial cell proliferation in an MTT assay( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) would be a substance or compound known to promote cell proliferation. One commonly used positive control for endothelial cell proliferation is vascular endothelial growth factor (VEGF), which is a potent angiogenic factor that stimulates endothelial cell growth and proliferation. Other compounds that can be used as positive controls include basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF), which also have been shown to promote endothelial cell proliferation.It is important to use a positive control to ensure that the assay is functioning correctly and to provide a benchmark for comparison to experimental treatments.
While many different biochemical assays can serve as reliable indicators of cell proliferation, it's important to determine that cell numbers are actually increasing in both positive and negative control cultures. Simply observing a difference between positive and negative control cultures in a biochemical assay may not be a good indicator of cell proliferation because it's possible that the cells may be actually dying-off at different rates. Depending on the medium you are using, a good positive control for endothelial cell growth might be to simply supplement the medium with deoxythymidine and deoxycytidine (each at 1 μg/ml). Article Nucleoside requirements for the in vitro growth of bovine ao...
Vascular endothelial growth factor (VEGF), was originally described as an endothelial cell-specific mitogen, and the activities of Vascular endothelial growth factor are not limited to the vascular system, but it plays a role in many normal physiological functions and is used in many assays as a positive control. So I think it's a good choice.
vascular endothelial growth factor or VEGF can be used for positive control
Inhibition of Proliferation and Stimulation of Migration of Endothelial Cells by Secretoneurin In Vitro
- Christian M. Kähler,Rudolf Kirchmair,Gerhard Kaufmann,Stefan T. Kähler,Norbert Reinisch,Reiner Fischer-ColbrieRuth Hogue-Angeletti,Hans WinklerandChristian J. Wiedermann
Originally published1 May 1997https://doi.org/10.1161/01.ATV.17.5.932Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:932–939
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