Gene pulser electroporation buffer

An aliquot of the synchronized worms was spun down at 500 rcf for 2 min to provide approximately 250 worms (unless otherwise specified) in a volume of 5 μl after the centrifugation. Then 5 μl of worms were mixed with 40 μl of electroporation buffer (Gene Pulser Electroporation buffer, Biorad, Cat. No. 1652676) in 1.5 mL tubes, and allowed to incubate on ice for 5 min. An aliquot of 5 μl of purified dsRNA (10 μg/μl) was added to the worms just before the electroporation, mixed by pipetting, and transferred to 0.2 cm pre-chilled electroporation cuvettes (Biorad, Cat. No. 1652082). Animals were electroporated at 300 V for 10 ms (unless otherwise specified) by square-wave single pulse using a Bio-Rad Gene Pulser (BioRad, Cat. No. 1652660). Immediately after the electroporation, worms were washed with 1 mL of pre-chilled M9 buffer, transferred into 1.5 mL tubes and centrifuged for 2 min at 500 rcf. Supernatants were discarded and animals were then transferred to E. coli OP50 seeded plates and cultured at 20° for 48 h.

The detailed procedure was based on the protocol from the provider (Bio-Rad, Hercules, CA, USA). Briefly, NSCLC cells (5x10⁷ cells/mL) were transferred into conical tubes and centrifuged at 1200 rpm for 5 min. After centrifuging, the medium were removed and the cells were washed with 1X PBS, and centrifuged again at 1200 rpm for 5 min. Afterwards, the tubes were added Bio-Rad Gene Pulser electroporation buffer. After resuspending the cells, the desired N1-GFP or FOXO3a-GFP plasmid DNA, kindly provided Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center, Utrecht, Netherlands) and was reported previously [26 (link)] and control (pCMV-6) or RUNX3 expression vector (RUNX3-pCMV6-AC-GFP, obtained from OriGene Technologies, Inc. Rockville, MD, USA) at a final concentration of 10 μg/mL were added and the electroporation plate were put in the MXcell plate chamber and closed the lid. The electroporation conditions on the plates to deliver 150 V/5 ms square wave were adjusted until reaching the optimum. After electroporation was completed, the cells were transferred to a tissue culture plate. We typically transfer each 150 μL electroporation sample to a 6-well tissue culture plate containing 2 mL RPMI1640. Cells were incubated 48 h at 37°C, then treated with baicalein for an additional 24 h.

CNE2 cells (1×10⁷ cells/ml) were transferred into conical tubes and centrifuged at 1,200 rpm for 5 min. After centrifuging, the medium was removed and the cells were washed with 1X PBS, and centrifuged again at 1,200 rpm for 5 min. Afterwards, the PBS was aspirated and added to Bio-Rad Gene Pulser electroporation buffer. After resuspending the cells, the desired N1-GFP or FoxO3a-GFP plasmid DNA at a final concentration of 10 to 20 μg/ml was added and the electroporation plate was put in the MXcell plate chamber. The electroporation conditions on the plates to deliver 160 V/10 ms square wave were adjusted until reaching the optimum. The conditions were set and loaded onto the device Gene Pulser II Electroporation System (Bio-Rad, CA, USA). After electroporation was completed, the cells were transferred to a tissue culture plate. We typically transferred each 150 μl electroporation sample to a 6-well tissue culture plate containing 2 ml RPMI-1640. Cells were then incubated for 48 h at 37°C, then treated with curcumin for an additional 24 h.

Resuspended exosomes were diluted in the Gene Pulser electroporation buffer (Bio-Rad Laboratories, CA) in 1 : 1 ratio. 1 μmol of mouse miR-132 mimic (Ambion, NY) or inhibitor was added to 200 μl of exosome sample. The mixtures were transferred into cold 0.2 cm electroporation cuvettes and electroporated at 150 V/100 μF capacitance using a Gene Pulser II system (Bio-Rad Laboratories, CA) as described previously [18 (link)]. After removing the free-floating miRNA mimic, exosomes were reisolated using ultracentrifugation. The final pellet (exosome) was resuspended in PBS and stored at −80°C.

To ensure the effectiveness of the two hit drugs in JEV replication, SA14 (GenBank accession no. U14163) replicon cDNA clone in which the structural genes were replaced with the Renilla luciferase (Rluc) gene was employed to quantitatively evaluate the inhibitory effects (32 (link)). In vitro transcripts were synthesized from linearized JEV replicon using a T7 mMessage mMachine kit (Albion, Austin, TX) according to the manufacturer’s instructions. Huh-7 cells were electroporated with in vitro transcripts in 800 μl of Gene Pulser Electroporation buffer (Bio-Rad) for electroporation cuvettes with a 4-mm electrode gap and a Gene Pulser II (Bio-Rad) unit at settings of 250 V and 950 μF, pulsing 1 time. After electroporation, the cells were plated in DMEM supplemented with 10% FBS, and compounds were added to the medium when specified. At the indicated times postelectroporation, the cells were harvested, and luciferase activity was measured using the Rluc assay system (Promega, Madison, WI).

For delivery of Cy5-mRNA-GFP into GPE-86 cells via conventional BEP, we used the Bio-rad Gene Pulser Xcell Electroporation System and Gene Pulser electroporation buffer (Bio-Rad Laboratories, US) (Additional file 1: Supplementary Sect. 1.4).