Polyethylene Glycol Fusion for Hybridoma Production

Once a good immune response has developed in an animal and an appropriate screening procedure has been developed, the construction of hybridomas is ready to begin.Polyethylene glycol (PEG) is the fusing agent of choice for hybridoma production, allowing the rapid and manageable fusion of mammalian cells.PEG fuses the plasma membranes of adjacent myeloma and/or antibody-secreting cells, forming a single cell with two or more nuclei.This heterokaryon retains these nuclei until the nuclear membranes dissolve before mitosis.In this protocol, antibody-secreting cells are isolated from the appropriate lymphoid tissue (mouse spleen and lymph nodes), mixed with myeloma cells, centrifuged to generate good cell-to-cell contacts, and fused with PEG.The fused cells are then diluted into selective medium and plated in multiwell tissue culture dishes.Beginning 1 wk later, samples of the tissue culture supernatants are removed from wells that contain growing hybridomas and tested for the presence of the appropriate antibodies.Cells from positive wells are grown, single-cell-cloned, and frozen.A procedure for screening batches of PEG for efficacy before hybridoma fusion is also included. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.RECIPES: Please see the end of this protocol for recipes indicated by .Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.Reagents Culture medium (with and without 10% fetal bovine serum [FBS]) (for screening PEG samples) D-10 medium for hybridomas DMEM HAT fusion medium HT medium (HAT medium without aminopterin) Mouse spleen or lymph nodes, fresh (see Step 14) Harvest tissues according to Protocol: Harvesting Tissue from Immunized Mice, Rats, and Hamsters (Greenfield 2017).MRC-5 cells Myeloma cells (for screening PEG samples) PBS (50 mL/fusion) Polyethylene glycol (PEG) 1450, including samples for screening if needed Fusions require very little PEG, and thus good batches of this reagent will last a long time.Bad batches contain trace amounts of toxic chemicals.For PEG screening, see Steps 1-7.However, only the occasional batch of PEG is From the Antibodies collection, edited by Edward A. Greenfield.