Hey guys, I am trying to generate 'nephrospheres' from mouse kidneys, but I am uncertain if I am just getting cell clumps or if you think these are fairly representative of spheroids? As a follow-up is there a good technique that would be definitive for saying that I am generating a spheroid and not just seeing cell clumps? These pictures are cells in suspension in low-binding plates.
I agree they look like spheroids, compact with a relatively smooth edge. Cell lumps normally are easily broken up by a gentle shaking.
Hello
from my experience with neurosphere, your picture refers that this is true spheres, and you could dissociate the spheres in to single cells by trypsin or according to your protocol. grow dissociated spheroid cells again to get new spheres.
Dear All,
I never cultured nephrospheres before, but I did culture lots of different kinds of primary cells, including "spheres"-like cells. I agree with them on how to identify "spheroids". I am also wondering the first/top one looks more like a cell clump, and the bottom two are spheroids as Vasily said. By the way, did you seed a population of cells that were completely separated and no clumps? Thanks
hi all,
I am not expert on this :( but i guess we can test this many way logically ...lemme know if you think its correct or not. Also i am not sure how accepted these techniques are ....
1) Necrotic core in spheroid...detect that and see if they are more in spheroid than in clumps.....hypoxia activated genes cab be looked in spheroids vs clumps...one should see obvious difference....Not sure about any marker!!
2) Clumps can be separated/dissociated with few round of pipetting...
3) Try low concentration of trypsin and visualise detachment of clump or spheroid in real time...you should see some difference... :)
4) Staining the suspension cells with some vital permeable dye and let them form spheroid or clumps for some time (hrs/days not sure).....and look by facs and see if u see difference in dye intensity in FACS...if they are spheroid the cells in core should not divide...and at periphery cell should divide in equal rate.....but in clumps they should divide and loose the dye equally ...
hope this will help..
This seems to be neurosphere.
You might wait after 2 weeks, then sphere forms bigger, then we can conclude. This type of cell line forms sphere easily. Also, 100um size is also considerable.
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