I recommend Corning ultra-low attachment (ULA) products. If you're looking to count sphere formation it's easiest in 96-well format. If you're expanding them further, it is easier to use the ULA T25 flasks. 

Hello Saghar,

Best plate format to culture spheres is 6-well ULA plates. I will not suggest to use ULA-T25 and 96-well plates because cells don't make good spheroids in T25 and we have observed that through our experience. A 6-well plate is infact best to grow mammospheres.

For components, 

You can use KSFM media provided with growth supplement and use it for sphere formation after adding 20ng/ml EGF,  20ng/ml EGF, and 1% B-27 supplement. I would recommend not to use a 96 well plate, infact use 6 well plate and seed around 1000-5000 cells/well in 2ml media per well. Add 1-2 ml fresh media on 3rd day and  do your assays till maximum 7th day as after that the spheroids will become necrotic.You can seed the formed spheroids lateron in 96 well if you want to do any specific experiment. Still if need to do in 96-well, you can seed 500 cells/well of a 96 well plate. Avoid to change any media, just add some fresh media after 3rd day so that nutrients can be replenished. Still if you want to change media, change at 3rd day after centrifuging the plate at 1000 rpm for 5 min.

Cheers !

1) The cheapest method is to form sphere on the underside of a 100mm sterile petri plates by hanging drop method, microbiological petri dishes (glass or plastic) work just fine.

2) Have sterile PBS as a source of hydration inside the plate.

3) Make 3-5 dilutions of cells in media at  5 cells, 10 cells, 15 cells etc per microliter.

4) Take a sterile plate lid and spot 25 microliter volumes from all the dilutions above.

5) Invert the lid so that your spots now "hang" with cells in them.

6) Incubate for 24 hrs or 48 hours without disturbing the plates.

7) Re-invert the plate and observe on a tissue culture microscope.

You need to understand the context of your question as well. If you want to use growth factors as a way of generating sphere then the method I suggested is not appropriate.

If you want to study sphere formation ability without growth factors then what I said before is a basic step.

I think you'll develop a lot more understand when you try out protocols. Good luck.

Can anyone provide a detailed sphere formation assay protocol for the prostate cancer cell lines? - ResearchGate. Available from: https://www.researchgate.net/post/Can_anyone_provide_a_detailed_sphere_formation_assay_protocol_for_the_prostate_cancer_cell_lines [accessed Jun 19, 2016].