Improving electrotransfection efficiency by post-pulse centrifugation.
We have demonstrated that the viability of electrotransfected adherent CHO and suspended NK-L, K-562, L1210 and MC2 cells is improved if pelleting by centrifugation is performed immediately after pulsing. The protection effect on cell viability is cell line- and pellet thickness-dependent. For forming CHO cell pellets, centrifugation force (300-13,000 g) and duration are not crucial; about five to 10 cell layers in the pellet provide the optimal protection effect. NK-L, K-562, L1210 and MC2 cell pellets are optimally formed by centrifugation at 13,000 g in an Eppendorf desktop centrifuge. Pelleting improves the cell viability over the whole range of the NK-L, K-562, L1210 and MC2 cell concentrations studied. When this pelleting method is applied to load CHO cells with FITC-dextran (41,000 MW), not only is the success rate close to 100%, but the growth rate is similar to the control, which is far better than the conventional electroporation method. Furthermore, the transfection efficiency of the five cell lines in pellet is significantly higher than that in suspension.
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Membrane Biophysics Laboratory, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Ross
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Hui
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D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002470:Cell Survival; D002498:Centrifugation; D006224:Cricetinae; D018274:Electroporation; D015870:Gene Expression; D007167:Immunotherapy / Q000379:methods*; D007694:Killer Cells, Natural; D008858:Microscopy, Phase-Contrast; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase