Improving electrotransfection efficiency by post-pulse centrifugation

Abstract

We have demonstrated that the viability of electrodorf desktop centrifuge. Pelleting improves the cell viability transfected adherent CHO and suspended NK-L, K-562, over the whole range of the NK-L, K-562, L1210 and MC2 L1210 and MC2 cells is improved if pelleting by centrifugcell concentrations studied. When this pelleting method is ation is performed immediately after pulsing. The protecapplied to load CHO cells with FITC-dextran (41 000 MW), tion effect on cell viability is cell line-and pellet thicknessnot only is the success rate close to 100%, but the growth dependent. For forming CHO cell pellets, centrifugation rate is similar to the control, which is far better than the force (300-13 000 g) and duration are not crucial; about conventional electroporation method. Furthermore, the five to 10 cell layers in the pellet provide the optimal protectransfection efficiency of the five cell lines in pellet is sigtion effect. NK-L, K-562, L1210 and MC2 cell pellets are nificantly higher than that in suspension. optimally formed by centrifugation at 13 000 g in an Eppen-

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses

“…This work describes the optimization of an electroporation protocol for K562 cells, involving Cell viability (% viable cells) was assessed by Trypan blue exclusion and transfection efficiency (% EGFP-positive cells) by flow cytometry the combination of electric field, resistance and capacitance values, combining standard electroporation processes with the post-pulse manipulation described by Li et al (1999). The egfp reporter gene has been widely used to analyze the efficiency of gene transfer protocols, and the pEGFP-N1 plasmid was previously shown to transfect K562 cells with use of the polycationic compound SuperFect (Qiagen) (Teixeira et al 2001).…”

“…After receiving the electric pulse, the cells were treated according to the protocol suggested by Li et al (1999). Briefly, cells were centrifuged at 13,000g for 30 s and maintained pelleted for 20 min.…”

“…Different parameters are analyzed, such as the conductivity and osmolarity of the electroporation buffer (Pucihar et al 2001), the cell cycle stage at which target cells are during electroporation (Takahashi et al 1991;Golzio et al 2002), or the effects of centrifugation immediately after the electric pulse is applied (Li et al 1999). Particular care must be taken with the viability of the transfected cells, since parameters, which increase transfection efficiency generally result in higher cell death rates.…”

Electroporation-enhanced gene delivery in mammary tumors

“…Lower transfection efficiency for samples with higher plasmid concentration at the optimal field strength may indicate membrane damage by DNA transport during electroporation. 13 …”

“…For electrotransfection, 15 l of cells at a concentration of 5 × 10 7 cells/ml in B+K buffer were mixed with uncomplexed plasmids in a custom made electrode chamber. 13 Cells were pulsed six times with 1 ms pulses at specified field strengths. Immediately after pulsing, cells were resuspended in 100 l of medium and incubated for 20 min at 37°C.…”