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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Chamber Cleaning
The chamber must be clean for two reasons; to minimize the
chance for contamination and to eliminate ions that increase
conductivity of medium placed in the chamber. (See the chamber
cleaning instructions on page 26) There have been many cases
where cell fusion has failed due to improperly cleaned chambers.
The chamber comes with two plastic caps. One size fits the top
snugly the other loosely. It is a matter of laboratory preference
which one to use. However, the caps should be kept on the
chamber when they are not in use and must be kept on the
chambers when the waveforms are being applied. The chamber
electrodes will have as much as 1000 volts between them.
Washes and Other Preparatory Needs
It is important that all tissue culture medium be washed from
the cells prior to electrofusion. This requires a minimum of one
centrifugation plus two washes in Cytofusion medium. Residual
ions from an incomplete wash will dramatically reduce hybridoma
production.
For the cell washes, it may be useful to wash the two cell
populations separately. This has two advantages. One is that if
there is differential cell loss during the washes, compensation can
be made during the mixing of the cells after the last wash. For
instance, myeloma cells are larger than B cells and may centrifuge
differently. Another advantage of combining the cells after the
last wash is that the two cell populations may be stained with
fluorescent dyes to differentiate the two cells after fusion and
to identify fused cells. Stained cells must be washed in separate
washes to prevent bleeding of the dye from one population to the
other during the washes.
While most labs mix the cells in the last step, others obtain better
yields by mixing the cells before the first wash.
If the cells used in the electrofusion are immortalized tissue culture
cells (the myeloma cells), the process is most efficient when cells
in log growth phase are used. Primary cells may not divide in tissue
culture and therefore may not be obtained in log phase.
It is also advisable to do the washes immediately prior to
electrofusion. Electrofusion medium is non-toxic but fusion
efficiencies have been known to decrease if the total time in the
Cytofusion medium (pre and post fusion) exceeds 30 minutes.
However, 30 minutes is a sufficient amount of time to complete the
process.
Generally cells should be mixed in a 1:1 ratio. Any decrease in the
myeloma cells-to-B cell ratio may result in decreased efficiency. If
one cell type is particularly rare, such as antigen specific B cells,
then more myeloma cells can be added to increase the chance that
a B cell will fuse to a myeloma cell rather than another B cell. Keep
in mind though that this will reduce the total yield.
Cell Preparation Process
• Harvest cells.
• Optionally mix the cell populations at a ratio of 1:1
• Connect the chamber to the Hybrimune Waveform
Generator with the provided cable
• Wash cells 2 times (3 centrifugations) in 20 ml
Cytofusion Medium, if the cell pellet is large, a third
wash will be needed. It is recommended that the cells
be centrifuged at 400 x g for 6 to 8 minutes to assure
minimal cell loss during was steps
• Re-suspend the cells in Cytofusion medium at a cell
density of 10 million cells/ml or other desired
cell density
• Mix the cell populations at a ratio of 1:1 if not
already mixed
• Place cells in the electrofusion chamber
• Important: the fusion steps must be performed within
30 seconds (e.g., as soon as possible) of loading the
cells into the chamber or cells will settle to the bottom
and give poor alignment. If allowed to sit more than 30
seconds, gently pipette the cells to resuspend before
proceeding
• Place the cap on the chamber
• Start the previously loaded fusion protocol
Producing Fusion Products Using Hybrimune
™