I am looking into protocols for making hybridomas and notice that some people grow their myeloma cells in 8-azaguanine and other don’t. What is the purpose?

This question is part of the following Ask The Expert session:

Monoclonal Antibody Production and the Culturing of Mouse Hybridoma Cells.

Answered by:

Timothy Fawcett, Ph.D.

Company: BioTechnical Institute of Maryland

Job Title: Director

Answer

Some people grow their myeloma cells (Sp2/0, Sp2/0-ag14, NS-0 and NS-1 cells for example) in 8-azaguanine or 6-thioguanineThe reason for this is to ensure that the cell line is 8-azaguanine or 6-thioguanine and HGPRT negative. Azaguinine and 6-thioguanine are competitors of guanine. This phenotype is necessary for the myeloma cells to be sensitive to the aminopterin in HAT (hypoxanthine-aminopterin-thymidine). Aminopterin blocks the nucleotide synthetic pathway and the HGPRT negative phenotype means that additionally the myeloma cells can not produce nucleotides via the salvage pathway and therefore will die unless fused. I suggest that the use of 8-azaguanine or 6-thioguanine at least some of the time is important to ensure the correct myeloma phenotype is maintained and only fused cells ultimately survive.

Previous Question:

I am a manager of a monoclonal Mab core lab for the university of Colorado, we have a history of successful hybridoma production but recently we have had problems with getting hybridomas from a mouse that shows a strong signal. On the first screening we will get strong positives that would traditional result in good hybridomas, but recently I have seen the positives stop producing the Mab after the initial screen. Few make it to cloning but don’t produce Mab after that. Do you have any idea why this is happening? We’re using mice that have had at least 3-4 injections with the protein of interest and are screened by Elisa titration assay and by a western. A traditional fusion is done with PEG to SP2/0 cells and plated into 96well plates. They are normally screened in 7-10 days. Media is IMDM+15% FBS +HAT

Next Question:

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?

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