Hi everyone! I am currently working with mammosphere cultures from both MB231 and MCF7 cell lines, and I have been struggling for quite a while. My aim is to have a method that assessed the drug effect on these cultures. As they are suspension cells and required these ultra-low attachment plates, this reduces a bit the choice, but I wonder what the best choice could be. I have tried trypan blue exclusion ( a no-go if we are testing several different conditions), Cell titer Blue (but as it requires black plates, i would need to transfer the cells into those, adding to variability), Acid phosphatase ( requires that media is replced by the reagent, so i don't think it is feasible), and lately, I have tried WST-1, but i am a bit unsure regarding it.
On another hand, I tired myself of reading articles of people that quantify mammospheres somehow, although this is never explicit on the protocols... Some use ImageJ, but never disclosed what properties they analyzed, neither how they treated the images. Others say they take pictures, and calculate based on size. Thing about this latter one is that these mammospheres cluster, so, if one has a 96 well plate setting, i can understand that with one picture one may be able to quantify it, but sometimes i see people using a 6-well setup, making impossible to quantify them all as one would have to take maybe 10 pictures/well... and if people claim they have triplicates of several conditions, then i am suspicious...
So, if someone is hands-on these type of things, please let me know... I would like to know more to improve my study!
Best regards
Tiago Braga
We basically counted them by eye in 6 well plates and measured from images. Yes, it was time-consuming and yes, we took lots of pictures! This was a while ago though and there may well be more automated methodologies in place now. The trouble is, I feel it needs some sort of visual input to distinguish between mammospheres and clumps.
https://www.researchgate.net/publication/5316477_Mammosphere_culture_of_metastatic_breast_cancer_cells_enriches_for_tumorigenic_breast_cancer_cells/rgformat
Article Mammosphere culture of metastatic breast cancer cells enrich...
You might wanna try Alamar Blue/TOX8 (resazurin-based, can be purchased from multiple manufacturers, but they always call it in their own way). It usually works well for 3D cultures.
Another approach might be CellTiter Glo designed for 3D cultures from Promega. It is an ATP-based assay. It's basically the old CellTiter Glo, but now they add more detergent in order to make disruption of cell clumps easier.
Both of them work on add-incubate-read basis. It's a bit difficult to understand what exactly you want to measure and why, but I hope this helps.
If your end-point is proliferation and/or apoptosis , Try Click-IT EDU fluorescent assays from Invitrogen. It works for microscopy or flow cytometry. The assay is very easy (no antibodies needed) and process to read-out takes less than 2 hrs. If the spheres are big you may want to use Accutase for dissociation and flow cytometry for counting.
Tiago, do not be suspicious about things that you do not know. It is possible and very easy indeed to quantify sphere using 6 wells plates.
Perform tumorsphere assay in 6 wells plates work fine. We do the follow:
First, to avoid aggregation as you had we use a medium containing methylcellulose (1%). With the methylcellulose the media will be thicker and the cells/spheres will not move much.
Second, we plate several different quantity of cells to start the assay (500, 1000, 5000 and 20000). With 500 we will find more variability and with 20000 more aggregation. So, we now are plating always 5000 cells.
Third, we use a plate reader to take 69 pictures per well in triplicate (yes, that is possible). We use the imageXpress software to take the pictures and them to analyse it. Of course that it is not easy at the beginning since you will need to set up and teach the software to analyse. Nonetheless, after this hard work the process will be relatively quick and automatised. Relatively because it takes around 16 min per plate to get the pictures and around 19 min per plate to analyse.
That is what we do to reach our goal. You will need to adapt to yours. For example, we do not assess effect of drugs as you do. I am saying that because if you need to add the drugs after few days of tumorphere culture you will face a problem to mix the drug with the methylcellulose media...
Hope that this answer will help you and did not come to late...
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