Cell Fusion - Fusing spleen cells with myeloma cells using PEG (Aug/10/2005 )
What are the best conditions for fusion using PEG?
Also, how SC-MC (spleen cells containing antibody secreting B-cells fuse with myeloma cells) hybridomas are selected for and other cells fusion products selected against by the use of HAT medium?
Hope someone can answer my questions. Thanks!
I have found that the literature in this regard to be very confusing with many conflicting recommendations so I'm not sure I can help you much with a step by step protocol-evryone swears by a different technique and I haven't had enough experience with it to say which is best. I will say that careful attention to keeping all solutions at 37C throughout the fusion is important and avoid sheer forces on the newly fused cells-no multichannel pipettes!
The HAT selection will select cells that have fused but does not discriminate between Ab producers and non-producers. Fusion cells that grow out need to be screened by ELISA (or a similar technique) and around 1-5% of these will produce Ab. Although I have read a paper where they claimed a 20% success rate.