i have performed MTT as per Mosmann T on HepG2 cell line using different concentrations of plant extracts (1-90 ug/ml). The absorbance of all the treated groups are reduced to about half as compared to the control (untreated) wells. However, the absorbance is fluctuating instead of decreasing with increasing concentration of the extract. what could be the reason? and what can i do to solve this problem???
It would be helpful if you could provide some actual numbers, please.
10,000 cells per 100 ul were seeded in a 96 well plate
0.271667
0.256933
0.2613
0.276667
0.254467
0.274067
0.243833
0.226433
0.2447
0.241267
0.276067
from up to down is the absorbance of increasing concentration..... the absorbance is an average of three readings...... sir adam
Do you have a positive control, meaning a result for a substance that is known to inhibit cell growth or kill cells? The data you showed looks like all approximately equal values, with no trend to indicate inhibition by the plant extract.
What s the solution of the extract and the absorbency spectrum of the plant material? These could be creating issues. One other confounding problem may be the uniformaty with your starting material. Does it include Chloroplasts? Could I see your MTT assay protocol?
HI
May be cell number per well too high. Decrease cell density at plating.
Secondly contamination of culture with bacteria or yeast. Discard. View wells prior to addition of MTT Reagent to check for contamination
Use positive control to check your results.
Good luck
Thiol-containing compounds and possibly other reductants can react with MTT giving a 'false' positive signal, therefore part of the absorbance signal could arise from components in your plant extracts. Perhaps try treating the cells with your extracts for a predetermined period (sufficient to see an effect), then washing the cells to remove the plant extract prior to evaluating the cell's survival with the MTT assay?
Hi Lalrinzuali,
I had the same problem when I started analyzing my compounds (some of them containing thiol and interfered in the signal, as Dr. Penketh commented previously). I solve it washing wells with PBS 1X after treatment to eliminate drug completely (wash wells at least twice), then add fresh medium and MTT solution (at the concentration requiered).
Hope it helps you.
Dear Lalrinzuali,
I had a problem with MTT when performing an exposure with coloured plant extracts and chemotherapic drugs, which were interfering in the absorbance reading. When I tried to wash the wells of the plate with PBS, to the complete removal of the substances, I lost cells and this caused another variable.
So, I've changed the assay by the resazurin test (or Alamar Blue assay), which can be based in the reading of the fluorescence emitted instead of using the absorbance as a the measurement criteria.
It worked pretty well to me and solved my problems.
Hope this information could help you.
Best regards.
If your compound is reducing compound then you might need to do the blank test keeping only compound in medium (without cell). This will give you the idea weather you can use MTT assay or not. If your compound is not reducing compound and still result is fluctuating then there might be problem in number of cell in each well plate.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line