Mammalian Heterokaryon Formation
• Tissue culture equipment
• Trypsin
• 18mm2 glass cover slips
• Appropiate tissue culture media
• 100 mg/ml cyclohexamide
• Phosphate buffered saline (PBS)
• Polyethylene glycol 5000
• Immunofluorescence materials
• Fluorescence microscope
Method
1. Grow mammalian cells of one species to subconfluent levels
2. Harvest the cells using trypsinization and seed the cells onto 18mm2 glass cover slips
3. Once the cells are attached, seed an equal number of mammalian cells of a different species onto the cover slips.
4. Incubate the co-cultures until the second set of mammalian cells have attached to the cover slip (approximately 3-4 hours)
5. Incubate the co-cultures with 100 mg/ml cyclohexamide for 30 minutes
6. Wash the cover slips with PBS
7. Fuse cells by inverting the cover slips onto a drop of prewarmed (at 37°C) polyethylene glycol 5000/PBS for 120 seconds
8. Wash the cover slips with PBS
9. Incubate the fused cells with 100 mg/ml cyclohexamide for 60 minutes
10. Fix the cells for immunofluorescence.
Alternatively, washes of the cultures can also be performed with buffers that are specific for the specific cell types.
Addition of 10 mM cytosinarabinoside to the fused co-cultures prevents overgrowth of non-fused cells.
[Borer, 1989; Pinol-Roma, 1992; Michael, 1997; Katahira, 1999]
