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Optimization of nucleic acid delivery into immune cell lines using Ingenio™ Electroporation Solution
Anjana N. Bhattacharya, Ph.D., and B. Gopalakrishnan, Ph.D.
Sponsored,vendor-submitted protocol    Sponsored by Mirus Bio LLC    Published in November 2009 (p.25) DOI: 10.2144/000113279

Abstract

Most cell lines of immunological origin are propagated in suspension and are known to be refractory to traditional chemical transfection methods. Electrotransfection using Ingenio™ Electroporation Solution achieves high-efficiency delivery of plasmid DNA and siRNA into these “hard to transfect” cell lines (Table 1) while maintaining cell viability. Since each cell type responds differently to electric pulses, best electroporation results can be achieved by optimizing the following key parameters: nucleic acid concentration, pulse type, and pulse conditions. Ingenio Electroporation Solution is compatible with Lonza-amaxa® Nucleofector®, Bio-Rad, and BTX® electroporators. This protocol provides guidelines for the optimization of plasmid DNA or siRNA electroporation into immune cell lines using Ingenio Electroporation Solution and Bio-Rad Gene Pulser Xcell™ electroporator.

Materials

Immune cell lines (e.g., Jurkat E6-1, K562, HL-60, THP-1, and U-937)

Complete growth medium

Nucleic acid plasmid DNA or siRNA

Ingenio™ Electroporation Kit, 0.4 cm cuvettes (Mirus Bio LLC, MIR 50113, MIR 50116, or MIR 50119)

Bio-Rad Gene Pulser Xcell™ electroporator (Bio-Rad Laboratories, Hercules, CA, USA)

Sterile tubes

Tissue culture plates

Reporter or other assay as required

Electroporation pulse types

Figure 1. Example of plasmid DNA titration on GFP expression efficiency in K562 cells. K562 cells were electroporated with varying amounts of EGFP reporter construct in Ingenio Electroporation Solution keeping pulse conditions constant at 250 V and 950 μF with Bio-Rad GenePulser Xcell™ electroporator.

Exponential decay and square wave forms are the most commonly used pulse types for mammalian cell electroporation. Ingenio Electroporation Solution can be used with both exponential decay and square wave forms. Certain cell types transfect better with square wave pulses while others respond better to exponential decay. Therefore, empirical testing is required for each cell type. The following protocol includes optimization tips for fine-tuning pulse conditions for both exponential decay and square wave forms.

Electroporation Optimization protocol





Table 1. Optimized exponential decay pulse conditions that result in both high transfection efficiency of plasmid DNA and viability for several immune origin cell lines. 1. Seed cells at a density of 1–2 × 106 cells/mL the day before electroporation. Incubate the cells overnight.

2. Harvest cells for electroporation.

3. Determine the final electroporation volume required for all electroporations by multiplying the number of electroporations by 0.25 mL (electroporation volume per 0.4 cm cuvette).

4. Resuspend the cells in the final electroporation volume from Step 3 at a final cell density of 5–10 × 106 cells/mL.

5. Add DNA or siRNA directly to the cell suspension, avoiding the formation of bubbles. Determine optimal nucleic acid concentration by testing a concentration range of 5–50 g/mL plasmid DNA (Figure 1) or 50–250 nM siRNA in the final electroporation volume.

6. For testing several different pulse conditions, prepare an electroporation master mix for the same plasmid or siRNA being electroporated. Add 0.25 mL electroporation master mix to each 0.4 cm cuvette. Electroporate the cells at room temperature, as follows:

a. For exponential decay: Test a voltage range of 200–300 V and a capacitance range of 800–1000 F. First, keep the voltage constant at 220 V while varying capacitance in 100 F increments starting at 750 F. Following this, vary voltage in 10 V increments starting at 200 V, keeping capacitance constant at the value determined from the capacitance titration.

b. For square-wave form: For converting exponential decay conditions to square wave, use half the pulse length and increase the voltage by ~10%, while keeping capacitance the same. For further optimization, test 10 V increments of voltage around the theoretically calculated pulse voltage.

Note: Using Ingenio Electroporation Solution, the ideal resistance setting is: zero ohms (Ω), or ∞. If your electroporator requires a resistance setting other than zero, use the lowest possible resistance.

7. Transfer the electroporated cells into a tissue culture plate containing pre-warmed complete growth medium.

8. Incubate the electroporated cells in appropriate culture medium for 12–72 h or as required.

9. Harvest cells and perform a reporter or viability assay as required.

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