If you are referring to hybridoma clones, the following two factors can be important in the establishment of stable clones upon fusion and limiting dilution:

1. Presence of feeder cells in the wells into which the limiting dilution was performed

2. Supplementing the HAT medium with conditioned medium (say, 10% spent medium from mouse fibroblast cultures)

Also, supplementing the HAT medium with 20% FBS is recommended to stabilize the hybridoma clones initially.

Hi,

are you using additional medium factors like non-essential amino acids, mercaptoethanol, interleukin-6, additional glutamine?

Have you tested your foetal calls serum stock before fusion?

Have you tested the osmolarity of the media? It should be 300 mOsm/kg (this is solved when you use commercial media)

Hi,

You need use HAT medium to culture, 8-AG(8-azaguanine) to select  hybridoma cells and at least 3 generation of  subcloning using limiting dilution.