Neon electroporation device

HEK-293 cells were purchased from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplied with 10% FCS, 2% L-glutamine, 1% penicillin/streptomycin and 1% MEM NEAA at 37 °C and 5% CO₂. HEK-293 cells used for experiments were not cultured beyond the 20th passage. Cotransfection of luciferase reporter plasmids and pre-miR-125a was carried out using 100,000 HEK-293 cells and 1 μg of psiCHECK-2 plasmid containing either the 3′UTR of JAM-A or JAM-L, respectively. Transfections were conducted using the NEON electroporation device (Thermo Fisher, 1150V, 2 pulses, 20 msec). After 40 h, cells were harvested, and reporter gene activity was assessed using the Dual-Glo Luciferase Assay system (Promega), according to the manufacturer’s instructions, on a microplate reader (FilterMax F3, Molecular Devices). All experiments were performed in triplicate.

PBMCs were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 2 d before electroporation. Cells were centrifuged and washed once with PBS. An aliquot of 1 × 10⁶ cells was resuspended in 100 μl electroporation buffer T (Thermo Fisher Scientific), and 100 nM siRNA oligonucleotides corresponding to human PNP was added. Electroporation was performed at 2,150 V, 20-ms and 1 pulse settings for stimulated PBMCs using Neon electroporation device (Thermo Fisher Scientific). Immediately after electroporation, the cells were plated in a 12-well plate with 100 U ml^–1 IL-2 and were incubated at 37 °C.

Transfections were conducted using the NEON electroporation device (Life Technologies) according to the manufacturer’s protocol. When HUVEC reached 80-90% confluency, cells were detached and transfected with Ambion^® hsa-miR-125 premiR™, hsa-miR-125 mirVana™ miRNA inhibitor (miR-125 inhibitor; both Thermo Fisher) or respective siRNA (Dharmacon). Transfections were carried out at final concentrations of 50nM (premiR™) or 100nM (miR-125a inhibitor, siRNA). Electroporation for HUVEC was carried out using 1 pulse of 1350 Volt and 30 ms.
For monocytes, transient transfections of miRNA mimic (premiR™) and siRNAs were performed using the NEON electroporation device at a cell density of 2x10⁶ cells and a final concentration of 50nM premiR™ or 100nM siRNA per transfection. Transfection efficiency of miRNAs was determined by flow cytometry using Cy3-labeled premiR™ negative control (Thermo Fisher). Knockdown efficiency of siRNAs was evaluated by qRT-PCR or SDS-PAGE. Cell viability was assessed after electroporation with 50 nM premiR™ negative control using flow cytometry of propidium iodide stained cells. Viability of monocytes was between 70 to 83% and viability of HUVEC was between 81 to 89%.

The DNA was introduced into BSRT7 by lipofection using Lipofectamine 2000 reagent in accordance with the supplier’s instructions (Life Technologies). Twenty-four hours later, RNA was introduced into BSRT7 cells using a Neon electroporation device (Life Technologies). The cells (10⁶) were suspended in R buffer (Life Technologies) with 50 ng of reporter mRNA (and 1 μg of standard RNA). The conditions for optimal electroporation were determined for the BSRT7 cells as two 20 ms pulses of 1400 V. After electroporation, complete culture medium was immediately added, and the cells were incubated at 37°C.
At the indicated times, the cells were lysed in the passive lysis buffer (Promega) for 15 min and then frozen at -20°C. Luminescence was measured using the Dual-Luciferase-Reporter Assay System (Promega) and a luminometer (Sirius, Berthold). The results are from three experiments conducted in triplicate with three different reporter mRNA preparations. The results were analyzed using Student's two-tailed t-test.

Transfections were conducted using the NEON electroporation device (Life Technologies, Waltham, MA, USA). Transient transfection with microRNA precursors (premiR, Thermo Fisher Scientific) or siRNA (Dharmacon, Lafayette, CO, USA) was carried out at final concentrations of 50 nM (premiR) or 100 nM (siRNA), and 250,000 cells per well. Cells were incubated for 36 h at 37 °C and 5% CO₂ in an antibiotics-free medium. Stable transfection was performed using 1 million cells and 10 µg plasmid/well. After incubation for 12 h in antibiotics-free medium, cells were seeded in DMEM containing 750 µg/mL Geneticin (Life Technologies). Stable transfection was analyzed by flow cytometry (Attune, Life Technologies). Monoclonal cell lines were obtained by single-cell picking. Overexpression of miR-744 was assessed through TaqMan assays. Co-transfection of luciferase reporter plasmids and premiR™ was carried out using 100,000 HEK-293 cells and 1 µg of Psi-CHECK™2 plasmid. All transfection experiments were performed in triplicate.