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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Immediate Post-Fusion Care of Cells
After the electrofusion process, cells begin to completely fuse. Even
though the pores that are responsible for the fusion were formed in
a few microseconds, the full fusion of cell membranes takes many
minutes. In a practical sense, this fusion maturation occurs over
the next 30 minutes. During this time, the cells should be handled
gently. The initial pores are very small (nanometers). While the
pores are small, they are fragile. As the cell membranes finish their
fusion, the fused pores expand to the size of the cell. The following
is a recommended procedure:
• Allow the cells to rest 5 minutes undisturbed.
• Harvest cells from the chamber using a sterile
disposable transfer pipette.
• Flush chamber with culture medium to remove cells
that have settled to bottom.
• Add cells to 50 ml conical tube and fill tube to 40 ml
mark with complete culture medium.
• Allow cells to rest an additional 25 minutes.
• Dilute the cells to an appropriate volume for plating.
• The cells can be immediately plated and cultured
without washing.
Materials Needed for Post Electrofusion
Culture
Cloning Medium 1
• DMEM
• 20% Fetal Bovine Serum
• 10% Origen Supplement
(Note: this supplement is no longer commercially
available. A filtered supernatant from J774A.1 cells
can substitute)
• 1% OPI Supplement (Sigma O5003)
• Penicillin-Streptomycin 1 X
• Sodium pyruvate, 110 mg/L
• Glutamax
• 50 µM 2-mercaptoethanol
• HAT diluted to 1X
Cloning Medium 2
• RPMI 1640
• HFCS (50X) diluted to 1X (Roche 11 363 735 001)
• HAT diluted to 1X
• 24 µM 2-mercaptoethanol
• Penicillin-Streptomycin 1 X
• Sodium pyruvate, 110 mg/L
• Glutamax
Complete Growth Medium
• DMEM or RPMI 1640
• 10% Fetal Bovine Serum
• Penicillin-Streptomycin 1 X
• 50 µM 2-mercaptoethanol
• Glutamax
• Sodium pyruvate, 110 mg/L
HAT Stock (50X)
• Hypoxanthine 5 mM
• Aminopterin 2 mM
• Thymidine 0.8 mM
HT Stock (50X)
• Hypoxanthine 5 mM
• Thymidine 0.8 mM
Cell Handling After Electrofusion
Producing Fusion Products Using Hybrimune
™