I would like to find out how many % of FBS and what media are used when you did your MTT assays with breast cancer stem cells.
Some said that they used 10% FBS with DMEM/12 and incubate in regular incubator which is 5% CO2 and 20% O2. Will this work? Can the result still be a representative for breast cancer stem cells?
Dear Jamail,
I would suggest using the conventional cell culture conditions you describe (10% FBS, 5%CO2, 37C) to start with, comparing the viability of cells with respect to treated populations (e.g. with your drug of choice). Once you optimise these conditions (e.g. amount of cells for a desired time frame, time you allow for the formazan production, etc.), then I would try and set up a more representative scenario. For instance, if you want to focus on the effect of a drug under hypoxic conditions, or a reduced serum content, I would use the previous protocol as a starting point to see how your new experimental conditions affect your specific cell population, and then evaluate and compare the effects of both cell culture and your drugs on cell viability. In this way, you will have a complete picture of the effect(s) of variables on your study.
Good luck.
Julio.
Dear Jamail,
I would suggest using the conventional cell culture conditions you describe (10% FBS, 5%CO2, 37C) to start with, comparing the viability of cells with respect to treated populations (e.g. with your drug of choice). Once you optimise these conditions (e.g. amount of cells for a desired time frame, time you allow for the formazan production, etc.), then I would try and set up a more representative scenario. For instance, if you want to focus on the effect of a drug under hypoxic conditions, or a reduced serum content, I would use the previous protocol as a starting point to see how your new experimental conditions affect your specific cell population, and then evaluate and compare the effects of both cell culture and your drugs on cell viability. In this way, you will have a complete picture of the effect(s) of variables on your study.
Good luck.
Julio.
These cells are routinely grown with DMEM supplemented with 10%
fetal calf serum and 1% penicillin/streptomycin, They
are grown on Flasks at 37°C in a
humidified atmosphere containing 5% CO2.
I would not recommend to use MTT assay with breast cancer stem cells. MTT assay is good for regular cancer cells that can form nice monolayer of cells on the bottom of the flask. Breast cancer Stem cells are not supposed to form monolayer, but they form in hollow clusters, mammospheres. The mammosphere formation assay should better reflect the growth capacity of these stem cells. you can find the mammosphere method in the attached paper.
Thanks guys. Actually, what I have found was stem cells can never be cultured with 10% FBS. If one cultured it with 10% FBS, it will differentiate and loss stemness. Since stem cells include only 1 to 3% in total population, I think it has to be very careful. Otherwise, I might be testing like a type of cancer cells rather than caner stem cells.
I have checked cell growth rate in hypoxic condition with serum free or 1% max serum. It was fine. I may just need to increase a bit seeding cells number. Since cells are in low or no serum, half of them are not attaching to the flask. Just a round cell and no differentiate. Some even form sphere in conventional flask or enhanced cell attach Nunclon delta flask too. I find out to use special dye that is not necessary to dissolve formazan crytals. So I can skip the step of removing media out. Removing media will give me a problem because unattached cells will also be gone and affect to absorbance value. I will try this way and my MMT result for this BCSC can be compared with MTT result from testing just cancer cells line.
Olga Sukocheva's suggested method is also great but counting the spheres will have problems. In the paper she attached, I noticed mammosphere formation test and also colony forming assay. What are the differences in term of aim of checking these assays? Do we need both or just one is enough? Is the aim of these tests something similar to in vivo tumorigenicity test? Also confuse about migration test or wound healing versus invasion test too.
You can determine viability and robustness of growth using the MTT assay with mammospheres. Just utilize nonadherent plates, and keep the FBS low enough so that you do not induce MET, the opposite of EMT. I have found that the stem cell characteristics of Breast cancer stem cells very easily fluctuate between a more epithelial phenotype and the classical mammoshere, this is one of the real challenges when working with cancer stem cells. Check out my MTT methods paper for ideas to maximize your assay.
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