Eppendorf Multiporator - Manual (Page 23)

65
4.8.7 Cleaning and disinfecting the Helix fusion chamber
The beaker and core of the Helix fusion chamber should be rinsed with
distilled water directly after the experiment in order to prevent cell and
buffer residue from drying up.
If heavily contaminated, the Helix fusion chamber should be cleaned
briefly in an ultrasonic bath (possibly with a cleaning supplement, such as
Edisonite Super) or with a very soft (tooth)brush. When cleaning is carried
out using brushes, please ensure that brushing is carried out in the same
direction as the windings, since the electrodes may otherwise move out of
their correct position and thus render the Helix fusion chamber unusable.
Disinfect the parts using non-denatured 70 % ethanol. To do this, the
beaker is filled with 250 µl of ethanol and the core screwed into the
beaker. After
10 seconds , the core is unscrewed and the alcohol can be
removed. To subsequently dry the beaker and the core place them in the
stand under sterile, dust-free conditions. After drying, the Helix fusion
chamber may be re-used.
4.8.8 Electrofusion buffer for eukaryotic cells
The electrofusion medium (fusion buffer) is of considerable importance for
the survival rate of the cells and for the successful extraction of hybrid
cells. Only a single buffer should be used for the entire electrofusion
procedure (alignment, fusion, post-alignment) in order to protect the cells
from additional stress. The fusion buffers from Eppendorf are
differentiated from the commonly used fusion media by low conductivity
and low osmolarity. The low conductivity (120 µS/cm) of the Eppendorf
buffers enables the application of relatively low field strengths (voltage)
when merging cells.
Ideally, electrofusion should be carried out in hypoosmolar buffer. As a
result of the hypoosmolar buffer system, the cells absorb water shortly
before the pulse and swell. The membrane and actin skeleton proteins are
thereby temporarily disengaged, thus easing fusion in the electrical field.
The yields of fusion products attained can thereby be considerably
increased in comparison to those under isoosmolar conditions. For cells
that react sensitively under purely hypoosmolar conditions, the necessary
osmolarity can be adjusted through step-by-step addition of the
isoosmolar buffer.
The Eppendorf fusion buffers (hypo and isoosmolar) are tested for sterility
and the absence of mycoplasma and endotoxins.
4 Device description
Multiporator_Text_en.fm Seite 65 Freitag, 9. Juni 2006 8:05 08