Monitor the centrifuge speed and time. Remove all of the supernatant from the pellet. Take particular care to keep all sterile tubes, solutions and pipets uncontaminated. Do not let water from the bottom of tubes drip into other tubes. Perform the following steps described below.
Timing is critical.
Use a stop watch to keep track of the time for each step. Whenever possible, perform the procedure over the entire time period, not merely in the first few moments of the time period. Add 20 ml of SFM over the remaining 4 minutes. As the volume increases in the original round bottom tube, transfer the contents into an empty 50 ml conical tube. The longer the cell solution remains in contact with the PEG, the more cells will fuse.