Bio-Rad Gene Pulser MXcell™ Electroporation System - Manual (Page 25)

Gene Pulser MXcell™ System Manual | Prepare Mammalian Cells
16
Harvesting and Counting the Cells
To harvest and count cells, follow these instructions:
1. Passaged the cells the day before electroporation. All cell types should be harvested
when they are actively growing. If working with adherent cells, trypsinize the cells to
detach them. Add growth media and then pellet the cells. If working with suspension
cells, pellet the cells.
2. After pelleting the cells, remove the media and wash the cells once with PBS by gently
pipeting them.
3. Remove an aliquot from the washed cells and count the cells.
Preparing the Cells for Electroporation
To prepare the cells for electroporation, follow these instructions:
1. Aliquot the number of cells needed to perform the experiment. For adherent cells, we
recommend using 1 x 10 6 cells/ml, but we have successfully used 0.5-5 x 10 6 cells/ml.
For suspension cells we recommend 2-3 x 10 6 cells/ml, but we have successfully
used 1-5 x 10 6 cells/ml.
2. Pellet the cells. Aspirate the PBS and resuspend the cells in the appropriate volume of
Gene Pulser electroporation buffer reagent (1 ml per 1 x 10 6 of adherent cells, and 1
ml per 2-3 x 10 6 of suspension cells).
3. Add the nucleic acid or other molecule. For siRNA electroporation, use 10-100 nM of
siRNA. For plasmid DNA electroporation, use 5-40 μg/ml.
Electroporation
To electroporate cells, follow these instructions:
1. Use 100-200 μl of mixture (cells in electroporation buffer reagent with nucleic acid) per
well of a 96-well electroporation plate. Use 500-800 μl of mixture per well of a 24-well
electroporation plate. Use 1-1.5 ml of mixture per well of a 12-well electroporation
plate.
WARNING! All wells in a well set must be filled with either sample or sample
buffer. For example, if you want to electroporate six wells, fill a complete well set
(such as ABCD1) with sample and fill two wells in a second well set (such as
AB2) with sample. Finally, be sure to fill the remaining two wells in the second
well set (such as CD2) with the sample buffer.
2. Rock the plate to wet the electrode and distribute the cells evenly
3. Transfer cells to tissue culture dishes containing growth media.
4. Incubate cells at 37 °C in a humidified CO
2
incubator until ready to be assayed.
5. After incubating 24 hours change the growth media.