When cells are transfected by Nucleofection®, transient pores are generated in the cell membrane. Generally, these pores disappear within 15 minutes of transfection, but if cells are plated immediately after Nucleofection® in medias that contain high levels of calcium ions (such as DMEM), then calcium ions can enter the cell, and activate calcium-sensitive pathways.

With stem cells, it is certainly possible that the influx of calcium ions into these cells can affect them adversely. There are many signal transduction pathways that are activated by calcium influxes, and in some stem cell types, part of the various differentiation signals involve calcium influxes. We have also seen with other cell types that activation of signal transduction pathways immediately following Nucleofection® can cause increased cell death, and this could conceivably happen with stem cells as well.

This potential problem can be eliminated by using a recovery step with a calcium-free media. After Nucleofection, add 500 µl of a calcium-free version of your growth media (with or without serum) to the cuvette containing the nucleofected cells, carefully remove the cell suspension, place it into a microcentrifuge tube, and then place the tube at 37°C (either in the incubator or in a heat block). After 10-15 minutes, the pores generated during Nucleofection® are closing or have already closed, and the cells can be safely placed in the normal growth media as usual. Since the pores are mostly closed by this time, the presence of calcium ions in the media will not affect the cells. Although our preference is to use a calcium-free version of your normal growth media, many people have successfully used a low-calcium media such as RPMI instead, and this remains a readily-available alternative "recovery media" for this purpose.