Hi. I'm studying in 3d cell culture.
Recently i stained my MCF-7 spheroid in GelMA scaffold, but the result is little bit strange.
I understand that big spehroid(left top[300 micronmeter]) has live and dead cell at the same time.
However, it seemed to be the same in small one.
I used live/dead cell imaging kit from invitrogen and fluorescence microscope(not CLSM).
To decide about overlapping, you should be able detect individual cells. At this magnification, it is not possible.
Next time I would suggest performing single channel controls on two samples - one just with live stain and one just with dead. By imaging in both the red and green channels you can determine if you are picking up red signal in the green channel and vice-versa. This will help you set microscope exposure times.
And like Attila said, higher resolution will help you determine individual live and dead cells.
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