should the low attachment plate that is used for culture be marked as grids to facilitate the counting of mammospheres manually? or does it require any software assistance? since I carry out this mammosphere assay for the first time in my lab, I have no idea as to how those mammopspheres formed in suspension cultures should be counted in order to calculate the mammosphere forming efficiancy (MFE). Please give me some ideas. Thanks in advance .
Hi jennifer,
Here are two possible options:
1. You could count the mammopheres directly in the growth plate. Follow dillution techniques if your intial density is high. Your output could be calculated as:
Number of spheroids/ml
2. Cell block preparation: After gentle spin (check literature), collect the spheroid cell pellet and make a cell block (firin clot method), cut at 5-8um thick stain with H&E and count spheroids under the microscope. Units could be: spheroids/cubic mm
Hope this helps
Parimi
Hi Jennifer,
I agree with Parini. I usually count directly in the growth culture using microscope. To evaluate the sphere:-
1. Count spheroid number.
2. Measure the size of spheroid.
In my case, i usually calculate both spheroid number and the size. Since this assay take longer time to develop sphere, you can measure each spheroid at day-1, day-5 or etc.
Hello
i agree with all researcher , look at the papers in links here
Good Luck
http://onlinelibrary.wiley.com/doi/10.1002/stem.2219/pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073404/
http://www.jove.com/video/52671/mammosphere-formation-assay-from-human-breast-cancer-tissues-cell
http://www.ncbi.nlm.nih.gov/pubmed/22665270
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428520/
http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc01e07s20/pdf
Hi all,
Thank you so much for your ideas and reference articles. I would try the above mentioned methods you have suggested.
An automated counting option may be helpful to you here. The GelCount, for example, can unbiasly count these spheroids and remove the step of counting under a microscope. This option would provide a precise measurement. Cheers.
Here is a webinar on some of the merits of automated colony counting.
https://www.scintica.com/webinar-automated-colony-assay-counting-advancing-the-frontiers-of-cancer-research/
And if you'd like to read a few of the publications a list is provided here.
https://www.scintica.com/wp-content/uploads/2018/03/Oxford-Optronix_GelCount_Citations.pdf
Its cool! thank you so much for letting me know this updated method for sphere counting!
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