I am using a series of bioassays to evaluate the biological response of different treatment processes. All of my effluents are extracted and I am evaluating the effluents as a whole, not according to different compounds present in them. The other bioassays I am using already have known and popular positive controls so I am able to calculate TEC. It was also suggested to calculate TEC for MTT & NRU if possible instead of only IC50.
I'm currently been searching for a positive control to for these assays (MTT & NRU) to have a comparison with samples and create a TEC value. So far in my research, I cannot find a standard one, particularly for HEPG2 cell lines.
Currently, I am interested in the cytotoxic mechanism of action compound as a positive control. I'm focusing on the mode of action of MTT related to cell activity and NRU as a cell proliferation measurement both calculated through cell viability.
I hope this helps and I look for more advice. I was thinking of using Phenol but I'm having a difficult time looking for a reference.
HI Lea Chua Ian,
Can you exactly brief us what your are looking for, either do you need a mitogen for cell proliferation or an anti-proliferative substance. Since MTT and Neutral red detects your cell viability, So kindly let us know what your are going to do
Hi
from my point of view about positive control it is better if the compound you are testing is analoge or derived from specific anticancer drug so you can use that drug as a positive control, it is better to use compound similar or close in structure to compare with
Best Wishes
Farooq
You may need to define whether you are interested in a cytostatic or a cytotoxic mechanism of action compound as a positive control. You could perhaps try terfenadine, tamoxifen, papaverine or paclitaxel if not already tried but give the cells 16-24 hour exposure to compound to see effects.
Hi, what about 1% Triton-x which we use in other cytotoxicity assays.
Hi Everyone,
Thank you for your advice.
As for the details, I am using a series of bioassays to evaluate the biological response of different treatment processes. All of my effluents are extracted and I am evaluating the effluents as a whole, not according to different compounds present in them. The other bioassays I am using already have known and popular positive controls so I am able to calculate TEC. It was also suggested to calculate TEC for MTT & NRU if possible instead of only IC50. So I am now trying to find a good positive control for MTT & NRU.
Currently, I am interested in the cytotoxic mechanism of action compound as a positive control. I'm focusing on the mode of action of MTT related to cell activity and NRU as a cell proliferation measurement both calculated through cell viability.
I hope this helps and I look for more advice. I was thinking of using Phenol but I'm having a difficult time looking for a reference.
Hi,
i would agree to Iain - without more details it is hard to advice anything. Do you expect an improved or decreased cell growth/cell activity? Hence, what should the compounds we might be thinking of do?
As for the MTT assay: be extremely careful with reductive compounds (like plant polyphenols, ascorbic acid) - they easily interfere with the readout, giving you false positive results. To my experience, alamar blue assay is easier to hande and gives the same idea of the cells activity.
Cheers,
Kristian
Hi Lea Tan:
The idea of positive control is to validate your MTT method is working or not working. Therefore, choosing only one drug as a positive control is fine for your MTT assay. Unless you want to challenge your drugs with the current chemotherapy drugs, then you may consider to purchase a few current chemotherapy drugs for liver cancer.
MTT is a very common method to evaluate drug in vitro. Since you are dealing with HepG2 cancer cell and you are interested in cytotoxic drug, it should be fine for you to use chemotherapeutic drug such as cisplatin. The mode of mechanism of cisplatin is to damage the DNA and induces apoptosis in cancer cells. I am sure this is the drug of your interest. It has been widely used in many types of cancers including liver cancer.
I tend to agree with Harita. You can use Sorafenib tosylate (Nexavar) or Doxorubicin (Adriamycin RDF/Adriamycin PFS), conventional cancer drugs. Many studies use them for HepG2 positive control.
I have used 5-Fu for HepG-2 cells in MTT assay, the exposure time could be 20-24 hrs.
Thank you everyone for all these recommendation. I will bring this up to my seniors and see which one would fit us best.
I' m agree with Harita too. In my experience with Hep-G2 cell line i use doxorubicin 0.004 mg/ml and after 24 i measure almost 50% of cytotoxicity with cristal violet assay.
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