Currently im doing the cell viability test for 24h, 48h and 72h. On the 24h plate, the reading appeared confusing same as 48h plate. I have 7 extracts concentrations, 0.001%, 0.01%, 0.1%, 2%, 4%, 8% and 10%. The purple colour appeared all except 2%, 4%, and 8%. Does anyone having this experienced before? what are the next step for me to conclude this confusing results? I really hope anyone could help and guide me on this matter. Thank you in advance.
Dear colleague,
From time to time (More often than we would like) all of us receive “confusing” results and from time to time we can not find the explanation for them. That is why we are doing re-search.
In order to find the answer and to solve the problem, you have to formulate better why your results are “confusing”. What kind of results did you expect and why?
Did you repeat the experiment? Did you use the adequate cell concentration? When the cells in the well are very dense (especially when the compound stimulates their proliferation) the monolayer can fall down during the MTT procedure and you will receive false data about high cytotoxicity. Did you check your plates also under microscope? Are you sure that cell culture you use is OK and there is no contamination? Are the examined samples and dilutions also OK?
The effect of the compounds on cell viability and proliferation very often is time- and concentration-dependent. It is completely possible that at two different concentrations (even close) the compounds affect in a different way cell viability and proliferation.
You can also increase the concentration of the compound tested (if possible, of course) and/or incubation period (96h?).
What about the solvent – how did you dissolve your compound? What is the effect of the solvent alone on cell viability and proliferation?
There is a cocktail of factors that can influence your results. If you have not enough experience with MTT test - In order to be sure that everything is technically OK you can perform MTT with the same cells but with a different and well known for you compound - just to check the system.
In such type of experiments it is very important to establish the best combination “compound tested – model system (cell culture) – method (cytotoxicity assay)”. Actually, this is the first and the most important step.
It is better to repeat the experiment performing both MTT test and some alternative cytotoxicity assay (neutral red uptake cytotoxicity assay and/or crystal violet staining …).
These methods have different molecular/cellular targets and mechanisms of action and it is always helpful to compare such results. You can also include additional cell culture as a model system in your experimental design because the “sell-specific answer” also exist.
Good luck!
Dear colleague,
From time to time (More often than we would like) all of us receive “confusing” results and from time to time we can not find the explanation for them. That is why we are doing re-search.
In order to find the answer and to solve the problem, you have to formulate better why your results are “confusing”. What kind of results did you expect and why?
Did you repeat the experiment? Did you use the adequate cell concentration? When the cells in the well are very dense (especially when the compound stimulates their proliferation) the monolayer can fall down during the MTT procedure and you will receive false data about high cytotoxicity. Did you check your plates also under microscope? Are you sure that cell culture you use is OK and there is no contamination? Are the examined samples and dilutions also OK?
The effect of the compounds on cell viability and proliferation very often is time- and concentration-dependent. It is completely possible that at two different concentrations (even close) the compounds affect in a different way cell viability and proliferation.
You can also increase the concentration of the compound tested (if possible, of course) and/or incubation period (96h?).
What about the solvent – how did you dissolve your compound? What is the effect of the solvent alone on cell viability and proliferation?
There is a cocktail of factors that can influence your results. If you have not enough experience with MTT test - In order to be sure that everything is technically OK you can perform MTT with the same cells but with a different and well known for you compound - just to check the system.
In such type of experiments it is very important to establish the best combination “compound tested – model system (cell culture) – method (cytotoxicity assay)”. Actually, this is the first and the most important step.
It is better to repeat the experiment performing both MTT test and some alternative cytotoxicity assay (neutral red uptake cytotoxicity assay and/or crystal violet staining …).
These methods have different molecular/cellular targets and mechanisms of action and it is always helpful to compare such results. You can also include additional cell culture as a model system in your experimental design because the “sell-specific answer” also exist.
Good luck!
Thanks for the response Dr. I will investigate throughly the given answer.
I think that Radostina is right, but one point you have to check your solutions and your pipette, it is usually the reason for problemas at this levels
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating