I seeded Ca9-22 cells on a 96-well plate. After 24 incubation I added different bacteria in different concentrations and incubate this overnight. The next day I take out the supernatant and added MTT and incubate this for 2 hours. After the 2 hours I removed the MTT and added DMSO. After the DMSO I measured the OD570 of each well. My question is: how can I analyze these results and which test can I use?
The plate reader/program you used to measure absorbance should analyze the data for you. Maybe you could ask someone from your lab to show you how they do it.
Also, some bacteria can metabolize MTT. Hence when looking at cell survival after bacterial infection, it might be better to not use MTT (as you might get super high values even when the cells are dead, due to MTT being metabolized by the bacteria in the well). We routinely use Neutral Red uptake to measure cell death in bacteria infected wells. The kit is cheap and it takes 10-30 minutes to finish the assay (as opposed to 2-4 hours for MTT).
Julie Joseph already gave you a good suggestion regarding the test.
For analysis you may use GraphPad Prism software if you have it in your lab. If not, it is possible to use Excel and make graph and calculations by yourself, it is quite easy. Usually people use Hill formula for fitting data and calculating EC50.
There are some good suggestions here: https://www.researchgate.net/post/How_can_IC_50_calculate_using_excel
You need to plot the results as bacterial concentration (in increasing concentration on x-axis) vs. optical absorbance (OD570 values on Y-axix). If you used more than one type of bacteria, you can do grouped analysis (one curve per bacteria type). If your bacteria is killing cells or inhibiting cell proliferation, the absorbance should go down with increase of bacteria concentration or vice versa when compared to the control (without bacteria). I hope this information helps. Good luck
One should be careful in removing MTT and adding DMSO so that we did not lose any cells.We centrifuged the plates before this step.Plotting graphs of OD and bacterial concentration will give a fair idea of toxicity
In my experiment. It turned out that bacteria could reduce the MTT as well.
It means that bacteria could attribute to purple crystal.
So no matter how many times that I wash cell with PBS, they can not be removed clearly. Do you have similar problem with that?
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