the special selection medium. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells (Quinlan and O'Kennedy 1994). Commercial preparations that result from the collection of media supporting the growth of cultured cells and contain growth factors are available that can be used in lieu of mouse-derived feeder cells. It is also possible to use murine bone marrow-derived macrophages as feeder cells (Hoffmann and others 1996).

Step 5: Cloning of Hybridoma Cell Lines by Limiting Dilution" or Expansion and Stabilization of Clones by Ascites Production

At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time. Cloning by "limiting dilution" at this time ensures that a majority of wells each contain at most a single clone. Considerable judgment is necessary at this stage to select hybridomas capable of expansion versus total loss of the cell fusion product due to underpopulation or inadequate in vitro growth at high dilution. In some instances, the secreted antibodies are toxic to fragile cells maintained in vitro. Optimizing the mouse ascites expansion method at this stage can save the cells. Also, it is the experience of many that a brief period of growth by the mouse ascites method produces cell lines that at later in vitro and in vivo stages show enhanced hardiness and optimal antibody production (Ishaque and Al-Rubeai 1998). Guidelines have been published to assist investigators in using the mouse ascites methods in these ways (Jackson and Fox 1995).



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