Cell suspensions electroporation

Three microliters of the donor DNA solution and 1 pL of the trap vector solution are blended with a 20 pL of competent JC8679 cells for electroporation see Note 10), and the mixture is transferred into an electroporation cuvette with a 0.1 cm electrode gap. After an electric pulse of 1.67 kV is applied, 1 mL of SOC medium is added to the suspension, and incubated at 37°C for 3 h with vigorous shaking. This process is applied to each gene one by one. 

ES cell preparation Trypsinize subconfluent ES cells to a single cell suspension as described in Subheading 3.2.2., centrifuge the cells, resuspend the pellet in 10-mL electroporation buffer, determine the cell density, centrifuge the cells again and resuspend the pellet with electroporation buffer to 2 x 107 cells/mL. 

Transfer 500 p,L of the cell suspension to a 4-mm electroporation cuvette (the remaining cell suspension can be used as non-transfected control cells or for other transfections see Note 9). 

Add up to 50 xg of caveolin-l-YFP encoding plasmid to the cell suspension in the electroporation cuvette (see Note 10). 

Transfect cells according to Subheading 3.4. In step 10, however, transfer the cell suspension to two electroporation cuvettes, 500 iL each. Then in step 11 add 50 jig HA-dyn2-WT and HA-dyn2-K44A to the two cuvettes, respectively. 

Figure 9.154 Detection of CAT activity in transformed rice and petunia protoplasts by HPLC. (a) Petunia leaf, (b) Petunia cell suspension, (c) rice leaf, and (d) rice cell suspension protoplasts electroporated at 500, 625, 825, and 625 V with an 860 fiF capacitor, respectively. Protoplasts were transformed by electroporation with 20 figl mL pDW2. Protein extracts were prepared and used in a 200 /xL CAT reaction mixture. Typically 12 fig of crude protein was used per reaction except for rice cell suspension protoplasts, where 20 fig was used. Products were isolated and 1 to 5 fiL of purified products was injected on top of the column. Peaks C, chloramphenicol E, solvent peak, ethyl acetate M, 1- and 3-monoacetoxy chloramphenicol. (From Davis et al., 1992.)...

Composition of electroporation buffer is an important factor affecting electroporation yields. Ionic strength of cell suspension medium needs control, which determines resistance of the cell suspension and resultant RC time constant of the field pulse. Medium supplemented with Ca and Mg in mM concentration range is found to promote efficiency of transformation and cell viability. Erythrocytes electroporated in isotonic buffer in the presence of EDTA or membrane specific drugs showed significant modification in hemolysis response to electroporation [33,34]. Use of square wave pulse removes the medium conductivity mediated effects on cell/tissue electroporation outcome. Generally, cells are pulsed in suspensions of sucrose, mannitol, or sorbitol. Electroporation as well as incubation of pulsed cells can be carried out in medium containing usual cell culture recipes. 

Due to recent trends in cellular therapy, the pharmaceutical companies have put emphasis on the use of ex vivo electroporation for both DNA and dmg delivery. Using the flow thm system, employing a pump that moves the cell suspension through an electroporation chamber where cells are porated. The entire operation is aimed to a closed system to minimize contamination and facilitate commercial scale cell-processing operations. 

Add 250 pL cell suspension to each cuvet. Mix gently and proceed with the electroporation. The settings for electroporation with the Bio-Rad Gene Pulser are as follows 960 pF 220 V. The settings using the BTX ECM 600 are as follows 1200 pF Resistance Timer R5 220 V (Note 15). 

Transfection (described for one kind of plasmid/ one electroporation) for cell count combine 0.2 ml cell suspension and... 

Suspend 5 x 10 cells in a 400 pL of EB. Just before electroporation, add 5 pg of the vector to the cell suspension and infuse into an electroporation cuvette (rrrNote 5 and 6). 

Following electroporation, incubate the cell suspension at 21°C for 15 min with 4 pL of healing buffer and then add the mixture of 10 mL of buffer with 10 mL of low-osmotic-strength buffer to the electroporated cell suspension (rrrNote 7). 

Rubinsky s group presented the first microfluidic device to electroporate a cell (Davalos et al., 2000 Huang and Rubinsky, 1999). Their devices consisted of three silicon chips bonded together to form two chambers, separated by a 1 pm thick silicon nitride membrane with a 2—10 pm diameter hole. Because silicon nitride is nonconductive, any electrical current flowing from the top chamber to the bottom chamber must pass through this microhole. A cell suspension was introduced into the top chamber, followed by the immobilization of one cell in the hole by lowering the pressure in the bottom chamber. 

In an electroporation cuvet mix 25 pg of linearized-targetmg vector with 10 cells (0.8 mL of cell suspension) Sit for 5 min and then electroporate at 960 pF, 250 V in a Bio-Rad gene pulser with a capacitance extender see Note 11) Gently mix to disperse the pH gradient caused by the electroporation and sit 10-15 mm at room temperature. 

Nearly any type of cell (prokaryotic or eukaryotic) can be transformed by the technique of electroporation. Protoplasts are first prepared by enzymatic or chemical disruption of the host-cell membrane polysaccharides. Next, the recombinant vector is introduced to the protoplast suspension residing in a very low ionic strength buffer (or distilled water). This DNA-protoplast suspension is then subjected to one or several 250-V pulses delivered from a cathode and anode placed directly into the solution. This applied voltage gradient will cause a certain population of the cells (—1010 per... 

Over the years, extensive research on cell electroporation has revealed that the efficiency of cell poration is governed by several factors, such as, physical, electrical, biological, and suspension medium [22-25,27]. Therefore, optimization of electroporation parameters for each type of cell and for a particular desired application becomes essential for successful outcome. There are several parameters that should be considered for optimization of electroporation outcome especially for in vitro electroporation experiments. 

Most adherent cell types can be efficiently transfected with lipid reagents. Even notoriously hard to transfect cells such as neurons, certain primary cells, and embryonic stem cells can be successfully transfected under optimized conditions with the appropriate reagents, presumably because siRNAs only need to be delivered to the cytoplasm (unlike DNA plasmids that must enter the nucleus for expression). For reasons that are not yet clear, suspension cells tend to be refractory to lipid-based transfection and typically require electroporation or viral-based methods for delivery of siRNA. While electroporation efficiently delivers naked siRNA directly to the cytosol for access to RISC, thereby bypassing the endosomal compartment, the extensive cell death that results and large amounts of siRNA required are prohibitive for broader-range applications. 

In tissues, the extracellular volume is small compared to the intracellular volume-completely the opposite of the situation when performing in vitro electroporation where the suspension medium volume by far exceeds the intracellular volume. This means that the threshold for electroporation is lowered [5]. It also means that the cell may tolerate permeabilization better, for example, since loss of ionic homeostasis may be less pronounced [6]. 

Use of a high field to activate a membrane enzyme was first reported by Witt et al. (25) in 1976. They used dc pulses of approximately 1 kV/cm and of 1-ms duration to induce ATP synthesis by the chloroplast ATPase. Following this initial work, there have been many reports on 1-kV/ cm dc field-induced ATP synthesis in different ATP synthetic systems (see the literature cited in references 13 and 14). The main conclusion from these studies is that an applied field-induced transmembrane potential can facilitate ATP release from the enzyme whether a PEF can affect enzyme turnover is not clear. Because 1-kV/ cm dc fields also cause severe Joule heating of a sample suspension, thermal effects cannot be easily avoided except when very short electric pulses (microseconds) are used. Thus, the method has limited utility for electroactivation experiments. The PEF method is, however, quite popular for the study of electroporation and electrofusion of cell membranes (see the chapter by J. Weaver in this volume), electroinsertion of membrane proteins (26), and electrotransfection of cells (27). 

Harvest cells and suspend in ice-cold SP buffer to a density of 5 X 10 cells/mL with 5 mg/mL flira-2-dextran and 1 mM Ca. Place 20 pL of the suspension in a 2-mm electroporation cuvette and pulse once with 850 V at 3 pF and 200 O in an electroporator (GenePulser, BioRad). The time constant should be approximately 0.6 ms see Note 10). 

Grow suspension or adherent cells in DMEM containing antibiotic-antimycotic, L-glutamine, and 10% FBS to confluency before the day of electroporation. 

Electroporate cells at a brief pulse of 750 V/cm and duration of 0.7-0.9 msec at room temperature. Cell porator settings should be an initial voltage of 300, the resistance switch at low position, and capacitances of 50-/xF for suspension cells and 60 //F for adherent cells. 

The electroporation conditions and parameters described here were optimized for a mouse lymphoma cell line (L5178YD10/R) that grows in suspension and two adherent cell lines, HeLa and a human fibroblast (HT-5). Approximately 80-90% cell viability and 85-95% electroporation efficiency were achieved using the above... 

Pipette the cells up and down and transfer 0.8 ml of the suspension to one electroporation cuvette, omitting air bubbles. Electroporate with one pulse of 500... 

Human fibroblasts can be transfected by electroporation. For transfection, 4 X 10 cells are resuspended in 500 //I PBS containing 20 /itg DNA. The suspension is placed in an electroporation cuvette (Bio-Rad Laboratories, UK) with a gap of 0.4 cm, and kept in ice for 10 min before electroporation. For the electroporation, a Gene-Pulser Electroporator (Bio-Rad Laboratories, UK) can be used with the following working parameters voltage, 380 V capacitance, 850 fjF. After the pulse, the cells are resuspended in normal culture medium and plated onto cover-slips in 24-weU plates at a density of 1.5 x 10 cellsAvell (Bonazzi et al., 2005). 

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