or cloning hybridomas via the limiting dilution method (1-2 cells per well), what are your thoughts on the following: (1) serum conc=20% (hybridoma qualified FBS) (2) PEC feeder cells-approx. 2000-5000 per well (3) conditioned medium I make conditioned medium from the myeloma fusion partner (P3X or SP2/0). Is there a better cond. medium to use? What experience, if any, do you have with rat-mouse hybridomas? Anything special I need to know about cloning, growing, maintaining these hybrid cells?

Answer

This is a good set of questions that are all related around how to isolate a single cell into the well of a 96 well dish. The goal of course is to get that single cell to grow into many. The problem is that initial single cell in that big well gets lonely and doesn’t get the proper signaling to promote cell division and survival. The odds are against its survival. To answer your specific questions… 1) I don’t think adding FBS above 10% is helpful and any excess above that may cause problems later when you try and drop the concentration down to 10% for growth at a higher cell density. Incidentally, I suggest moving away from serum altogether for many reasons including purification of the MAb from the growth medium. There are a lot of great serum-free and even protein-free hybridoma media out there such as CD-Hybridoma or Hybridoma SFM. 2) PEC-feeder cells are peritoneal exudate feeder cells and the idea is to put some PEC-cells in the wells along with your single cell you want to clone. This was/is a lot of work, it involves irradiation or a chemical means to stop the growth of the feeder layer when necessary. It turns out that IL-2 or/and iL-6 is what is needed. My suggestion is to supplement your cloned cells with IL-6 (known as a myeloma growth factor) or something like briclone an IL-6 enriched cloning medium. 3) I am a big fan of conditioned medium. Conditioned medium is partially used medium and it has some factors secreted by cells, but it still has some nutritional value. I use conditioned medium for cloning a single cell and I usually use a 50% fresh /50% conditioned media mixture. I also make the conditioned media using the same cells I want to use in fusion such as the SP2/0 cells or the NS0 cells.