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The purpose of cloning is to isolate a single cell that produces the desired antibody from the hundreds of other cells in a culture so that a monoclonal cell line can be established. Immediately after the fusion of spleen cells to myeloma cells, several hybrid cells, all producing different antibodies, are often growing in the same microtiter well. In addition, the fusion process leads to the formation of unstable hybrids which lose chromosomes in order to become stable. This process can lead to the formation of mutants which do not produce or secrete antibody. These mutants often overgrow the desired hybrids which are producing antibody. For these reasons, it is essential that once cultures are identified that are producing interesting antibodies, that they be cloned to isolate the single cells that are producing the desired antibody. The most common method of cloning hybridoma cells is to dilute the cell culture out, so that when plated in a microtiter plate, there is only one cell deposited in each well. This cell then divides and gives rise to a culture were all cells are producing antibody that is of the same specificity. Because each cell line has a different cloning efficiency, it is necessary to plate out the cells using several different cell concentrations. The standard "limiting dilution" cloning that the immunology core performs on each selected culture, is to plate out the cells in 96-well microtiter plates using the following calculated cell concentrations: 2.0, 1.0 and 0.5 cells/well. An enriched hybridoma media with growth factors is used to support the growth of single cell hybridomas. When colonies begin to appear, each well is looked at microscopically to determine the wells where it is most likely that only one cell gave rise to the growth of the new culture. The fees below reflect the subcloning, marking the plates for clonality before screening and all subsequent tissue culture for the expansion and freezing of hybridoma cell lines. Supernatants are collected from every culture cryopreserved for further study. FEES: Fusion subcloning, which involves cloning 6-8
cell lines twice is $2135. Additional subclonings of 6-8 lines is $1092
per subcloning. |
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