Hello,
I am trying to understand a co-culture experiment using different ratios of a cancer cell line and hematopoietic stem cell fraction in a mixed co culture. The ratios range from 1 to 1000 for the cancer cell line keeping the other at 1. The proliferation was checked at 24, 48 and 72 hours.
My specific questions are
How would you account for cell number changes from different populations of cells in the MTT reading.
What are all the controls that need to be set up?
If you were setting up this experiment, how would you go about it?
I would be happy if someone could break this down for me.
-Swetha
Hi Swetha,
If I understand the question properly this is how I would go by with the experimentation.
I don't know of a way to find the cell number changes from different populations using MTT, I would do either of the two following things.
1) Tag one of the population with a fluorescence protein (transfection) and use FACS.
2) Co culture the cells with a mesh in between so that you can use MTT separately on the two populations.
Personally I prefer XTT as it is faster.
Hi Swetha,
If I understand the question properly this is how I would go by with the experimentation.
I don't know of a way to find the cell number changes from different populations using MTT, I would do either of the two following things.
1) Tag one of the population with a fluorescence protein (transfection) and use FACS.
2) Co culture the cells with a mesh in between so that you can use MTT separately on the two populations.
Personally I prefer XTT as it is faster.
I agree with Sajjeev. MTT is not a suitable stain to use for cell number detereminations because it cannot discriminate between the different cell types. I would recommend making a probe (or buy one commercially) that will make one of the cell types express GFP and the other RFP or some other color. Then you will be able to count both cell types and their progeny using FACS.
Hi dear Swetha,
First of all MTT assay is not very sensetive technique, I may advise you to use Prestoblue.
I did co-culture test for vitalities and toxicities.
Regard your cancer cell is SCC and the stem cell is ST
Do tests for the folllowings
1- SCC alone
2- ST alone
3- SCC+ST (co-culture)
4- Negative control any of these (1, 2 or 3) cultured with formaline or ethanol.
I hope my answer was clear, feel free for any more clarifications, please?
Best wishes
Sarmad
Thank you Sajjeev Jagannathan , Christopher Lange and Sarmad Al-Sahaf . All of your answers have been helpful.
Using fluorescence and Flow cytometry analysis or tagging the different populations with gfp/rfp seems to be the best way to do such an experiment.
I did have a separate question here (not connected to the experiment). How would you account for copy number changes in the transfected plasmids? the intensities of the gfp/rfp changes and is there anyway to keep it uniform (incase these are tagged with other proteins)?
Sarmad Al-Sahaf - what is the concentration of the formalin/ethanol that you use for the negative control? is it the same for all the time points? 24, 48 and 72 hours?
Also, when you say co-culture, what are the things to keep in mind with regard to deciding the ratio incase it isnt a 1:1 coculture? the populations would have different growth rates, so how would you relate the effect you are seeing after MTT specifically to the cell influences? Does such an experiment really give you clear answers?
-Swetha
Ideally you would use a lentiviral vector containing your gfp/rfp insert package. This would get your package to be a stable insert into the host genome. From your flow cytometry you can measure stain intensity for each cell and then place them individually into wells for future growth.
Further to my GFP/RFP answers, copy number will only affect fluorescence intensity, but if a single stable transfection produces sufficient fluorescence for detection on your flow cytometer, then all you need to do is count the numbers of red and green cells as a function of time to get growth curves for each population in the co-culture.
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