Some cells are just very hard to create spheroids... Have had similar problems. We use 0.04% Methylcellulose (MC) to induce spheroids and works fairly well.

Prepare a 2% solution of ME in your complete medium by dissolving in a shaker overnight (MC takes a very long time to solubilize since it needs to first solvate with water...). When solubilized, filter to sterilize (

I use methyl cellulose for liver cancer cell lines and it works pretty well for me. The protocol is easy and the results are reproducible. I use the same protocol as Helder Andre. You can follow this publication that I have attached. The trick here is the number of cells that you seed. The protocol says 4000 cells but that eventually depends on the proliferation rate of your cell lines. I use 500 cells to seed and in a day the spheroids are 200um in diameter.

Thank you Mr Helder and Mr Wasim for your valuable suggestions.

If i am use low-adherence round-bottom plates will i be able to get uniform size spheriods? I need uniform sized spheriods because i has to perform CTG assay which needs uniformity size?

can you post the protocol for using cell titer glo for 3D? thank you.