Hi Delynn, I have used sorting to significantly reduce the overall time of obtaining clones. But your primary question in regard to the benefit of pre-sorting pure Bcells before fusion, is a good one. It does not help at ALL! I would not waste my time on it. In my opinion, the only benefit you will find in using sorting in the generation of hybridomas, is if you can label the antigen that was used for immunization. This allows you to sort only antigen specific Bcells or hybridomas. I used both schemes to speed up the process. For example, I used mostly recombinant proteins to immunize the mice, which meant that there was plenty of antigen to also FITC label, a very easy process that you can do yourself. I would first sort the spleen cells for antigen reactive Bcells prior to fusion. At the time you would normally do your first plate cloning, I took the stable hybridomas from mass culture and labeled them with my FITC-antigen conjugate. By doing this I generated clones from the first and only set of 96 well plates (easier if your sorter can do single well cloning!). This greatly reduces the amount of time to obtain clones. I also generated a method to screen for relative affinity to the labeled antigen based on temperature. Please contact me directly if you would like information on this protocol or have any further questions. Kind regards, Steve McClellan BioConsulting Concepts (www.bioconsultingconcepts.com) email steve@bioconsultingconcepts.com 2156204774
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