![background image](http://html.mh-extra.com/html/harvard-bioscience/btx-hybrimune/btx-hybrimune_user-manual_601890026.webp)
26
Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Demonstration Experiment to
Illustrate Turbulence
A simple demonstration can be done in the lab to show what
proper cell alignment looks like and what it looks like when it is not
working correctly.
• Wash 10 million myeloma cells twice in Cytofusion
Medium C.
• Set-up the Hybrimune System.
• Program a 70 V pre-pulse sine wave for a 10 second
duration (other parameters can be set at minimum).
• Place the cells in the two ml chamber, place the
chamber on an inverted microscope and connect the
chamber to the Hybrimune Waveform Generator.
• Click turn on HV Pulse then Start when the start bar
becomes active.
• Observe orderly alignment of cells.
• Next, add 50 ml PBS to the cells in the chamber.
• Click turn on HV Pulse then Start when the start bar
becomes active.
• Observe turbulence and that the current indicator
shows red bars lit.
Optimization
The protocol presented on page 43 is a good starting point for
producing hybridomas by electrofusion using the Hybrimune
System. The cells from each lab probably have a different optimum.
The factors that most dramatically affect fusion efficiency are:
• Pre-pulse sine wave amplitude and duration
• Pulse voltage and pulse width
The pre-pulse sine wave second step amplitude is 75 volts peak. If
there is excessive turbulence, it may be reduced slightly. Decrease
the duration until the turbulence cannot be visually detected.
Another function such as Ramp k may also improve performance.
Next try decreasing pulse voltages and then increase pulse width in
steps of 10 µs.
Unless the post-pulse sine wave is causing excessive movement of
the cells, changing that parameter will have minimal effect on the
results.
Chamber Cleaning
Chamber cleaning is necessary to remove biological contaminants
and ions. The electrofusion process is sensitive to the presence
of ions. Clean chambers are essential to prevent excess ion
contamination.
Cleaning Process
The following cleaning process is recommended:
• Immediately after use, rinse the chamber in reagent
grade water.
• Fill the chamber with 4% sodium hydroxide and soak
for 5 minutes.
• Empty the chamber.
• Rinse in reagent grade water for 10 seconds.
• Repeat rinse 10 times.
• Rinse once in 70% ethanol.
• Air dry.
Chemical Sterilization
• Fill chamber with 4% sodium hydroxide and soak for
10 minutes.
• Empty chamber and fill with 70% isopropanol or
ethanol and soak for 10 minutes.
• Empty chamber and fill chamber with Spor-Klenz®
(Steris), soak 10 minutes.
• Rinse thoroughly in sterile reagent grade water.
Spor-Klenz® is a registered trade mark of Steris, www.steris.com.
Other Sterilization
Alternatively, the chambers can be gas sterilized. Do not autoclave
the chambers.
Producing Fusion Products Using Hybrimune
™