- I did MTS proliferation test on my PEG based hydrogels. I seeded 8000 L929 cells in a 96 well plate in DMEM medium (containing phenol red). The next day I changed the medium and added hydrogels on top of the cells. After 24 incubation, I again refreshed the medium and added 20 uL MTS to each well (without removing the hydrogels), incubated for 4 hours and took the absorbance reading at 490 nm. For calculations, I used the formula: ((absorbance of each well - backgroud absorbance)/(absorbance of untreated cells-backgroud absorbance))∗100 where background absorbance is only medium + MTS incubated for 4 hours.
- I found that my sample wells show higher viability than my untreated wells. I don't think it is due to pipetting errors since the experiment was simultaneously repeated by another person who got similar results. Another possibility I considered was that my hydrogels somehow interact with the MTS. My substances are PEG based amines and epoxides. I could not find any information about whether these can interact with MTS. Also the hydrogels seem deeply stained after the MTS incubation but I feel this could just be the hydrogel absorbing some formazan formed, rather than some chemical reaction. Does anyone have any ideas about why I have such high viability values for my sample wells?
- The images show the data and the wells before and after removing the MTS solution to show that the transparent hydrogels become dark. Wells 6,8-10 had hydrogels in them.
Gian Vincent Canlas Dizon my concern there would be that I would lose cells that become attached to /embedded within the hydrogel structure and get a wrong viability reading.
I would like to share some of my thoughts:
- first of all I would simply test if there is an interaction between the hydrogel and the MTS by introducing a "control" where you add the hydrogel in a well without cells and treat it as if there were cells (add medium, replace medium etc.) to keep it as comparable as possible. When the MTS interacts/reacts with the hydrogel you will find a difference between your normal blank value and the blank+hydrogel control.
- secondly, an MTS assay is not a sole proliferation assay, it is a cell viability assay indicating the metabolic activity of the cells. So is it possible that the hydrogel layer on top is somehow favorable concerning metabolism etc. compared to the normal 2D culture? Is it possible that the cells grow into the hydrogel? All important things to consider.
Kerstin Frey thank you for your comments. I finally came to the same conclusion and plan to use controls with hydrogels and no cells for my next trial. Also you are right that it might be that the cells grow better on the hydrogels. I will update on the results after I do the next trial
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