This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Cell preparation and construction of heteronucleosomes
Materials and Instruments
Mouse cells Move Co-culture of cells For more product details, please visit Aladdin Scientific website.
Trypsin PBS
Glass Coverslips
1. 12~24 hours before fusion, spread mouse cells onto sterile glass coverslips so that they can fully attach and stretch on them.
2. 3 to 4 hours prior to fusion, trypsin-digested human cells are spread onto glass coverslips containing mouse cells, with the number of human cells equal to or slightly higher than the number of mouse cells, also for better heterologous fusion.
3. Incubate the co-culture at 37℃ for 2-3 hours to allow the human cells to attach to the coverslip without overstretching. At this time, actinomycin ketone (CHX; 50~100 μg/ml) can be added to prevent new protein synthesis.
Heterokaryosome formation
4. Gently rinse the coverslips with the co-cultures twice with pre-warmed Ca2+ and Mg2+ free PBS.
5. Pick up the coverslip with small tweezers, drain the excess PBS, and place it cell side down on a drop of pre-warmed PBS solution with 50% PEG.
6. Incubate the coverslip in PEG for exactly 2 minutes.
7. Pick up the coverslip, being careful not to let it slide (it can damage the cells), and rinse it thoroughly twice with PBS. This can be avoided by holding one end of the coverslip with a yellow pipette tip while lifting the coverslip from the other end with a small pair of tweezers.
8. Place the coverslip back into the culture medium (still containing the protein synthesis inhibitor, if desired) and incubate long enough for the actively shuttled proteins to equilibrate between the nuclei in the heterokaryosome after 2 to 4 hours of fusion.
9. Fix and permeabilize the cells at the end of the incubation.