Journal: Scientific Reports

Article Title: Cellular transfection using rapid decrease in hydrostatic pressure

doi: 10.1038/s41598-024-54463-5

Figure Lengend Snippet: Efficacy of transfection of ES cells using pressure-jump-poration. ( A ) Transfection rate for the pressures indicated compared to standard electroporation conditions following transfection. NDN: (−) DNA control; DNE: (+) DNA, no electroporation; E.STD: (+) DNA, (+) electroporation; ND80: (−) DNA at P = 80 MPa; 0–100: (+) DNA, (+) pressure treatment; 70SL: (+) DNA, (+) pressure treatment, but with slow (3 min) pressure release. All other samples were held at the indicated pressure for 30 s followed by acute depressurization. For each pressure condition n = 9 independent experiments with three replicates within each experiment were performed, and n = 5 independent experiments with three replicates within each experiment for electroporation conditions. Results shown ± SD. ( B ) ES cells on gelatin at 24 h following pressure treatment at 60 MPa (no selection). Scale bar denotes 150 μm. ( C ) Clonal ES cell colony on fibroblast bed layer following pressure treatment at 60 MPa with subsequent puromycin selection for 5 days. All pressurization experiments were performed at a concentration of 4000 cells/μL with treated cells plated at 10,000 cells/well in a 6 well plate; electroporation standards were plated similarly. Scale bar denotes 200 μm. Results shown ± SD. *Denotes significant enhancement at p < 0.01 over electroporation. Transfection plasmid shown in ( B ) and ( C ) expresses dTomato/puromycin.

Article Snippet: To determine the relative efficiency of PJP, results with ES cells were compared in identical solutions and DNA concentrations to an optimized electroporation standard (E STD: Biorad 240 V, 500 μF in 4 mm cuvette).

Techniques: Transfection, Electroporation, Control, Selection, Concentration Assay, Plasmid Preparation

Journal: Scientific Reports

Article Title: Cellular transfection using rapid decrease in hydrostatic pressure

doi: 10.1038/s41598-024-54463-5

Figure Lengend Snippet: Properties of pressure-treated cells. ( A ) Differences in pressure-mediated transfection efficiency in immortalized versus primary cells. Shown are relative transfection efficiencies of primary fibroblasts (blue) vs. mouse L-cells (red) by electroporation and pressure-mediated transfection. Pressurization was performed at of 4000 cells/μL with cells plated at 10,000 cells/well in a 6 well plate; electroporation standards plated similarly. For each pressure condition, n = 3 independent experiments with three replicates within each experiment were performed. Results shown ± SD. ( B – H ) Cellular features and morphology of pressure-treated R1 ES cells. Live citrine-expressing ES cells were treated with 1.8 μM (1 μg/mL) Hoechst 33342, 10 nM (5 ng/mL) TMRM and 0.75 μM (0.5 μg/mL) propidium iodide prior to pressure treatment. ( B ) Cells in the absence of pressure treatment; ( C ) Typical appearances of ES cells following 1 min of pressure treatment at 100 MPa (pressurization at 4000 cells/μL, cells were held for 1 h at 25,000 cells/well in the slide chamber). ( D ) A small subgroup of these cells become PI+ but retain cellular features. ( E ) A major portion of these PI+ cells go on to exhibit features of cellular destruction in the immediate (30 min) post-treatment period. ( F – H ) ES cells following 5 min at 100 MPa. The majority of these cells demonstrate features shown in ( F , G ). ( G ) A portion of recovered cells exhibit features of reduced cellular volume. ( H ) The great majority of cell which become PI+ following treatment at 100 MPa for 5 min exhibit features of cellular degeneration. For ( B – H ) scale bar indicate in ( B ) represents 10 μm. ( I – K ) Electron photomicrographs of ES cells. ( I ) Following rapid depressurization at 80 MPa, a population of ES demonstrated the presence of intracellular voids proximal to the cell membrane (red arrows), frequently associated with protuberances of the cell membrane (blue arrows). ES cell held at ambient pressure is shown for comparison ( J ). By contrast ES cells subjected to slow pressure release at 80 MPa, ( K ) often demonstrated extensive extrusions (blue arrowheads). For figures ( I – K ) scale bar represents distance of 1 μm.

Article Snippet: To determine the relative efficiency of PJP, results with ES cells were compared in identical solutions and DNA concentrations to an optimized electroporation standard (E STD: Biorad 240 V, 500 μF in 4 mm cuvette).

Techniques: Transfection, Electroporation, Expressing, Membrane, Comparison

Journal: Cell Journal (Yakhteh)

Article Title: Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

doi:

Figure Lengend Snippet: Physical map of the vectors and electroporation procedure. A. Both pVASA -EGFP and pOLIG2 -EGFP benefit from dual selection system including neomycin and puromycin. No difference was seen in transformation efficiency despite the different vectors’ sizes and B. Human embryonic stem cells (hESCs) were seeded onto 50 cm plates and grown in appropriate medium until they reached 70-80% confluency. Following incubation in Tryp/LE at 37˚C for 5 minutes the cells were singled. For electroporation, the cells were counted and resuspended in phosphate buffer saline (PBS) at a concentration of 1×10 6 , after which 700 μl of cell suspension was mixed with 20-60 μg of linear plasmid DNA in a sterile electroporation cuvette. The voltage varied from 240 to 300 V. Immediately after electroporation, the cells were removed from the cuvette and plated on three 10 cm diameter tissue culture dishes in complete medium. After 48 hours, the plates were washed twice with phosphate buffer saline (PBS), then replenished with complete medium. MEF; Mouse embryonic fibroblast.

Article Snippet: In brief, 600 μl of the previously singled cells that contained 10-60 μg linearized plasmid was transferred into the electroporation cuvette (BioRad, USA, #165-2088).

Techniques: Electroporation, Selection, Transformation Assay, Incubation, Saline, Concentration Assay, Suspension, Plasmid Preparation, Sterility

Journal: Cell Journal (Yakhteh)

Article Title: Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

doi:

Figure Lengend Snippet: In vitro transfection of H5, H6 and human foreskin fibroblast (hFF) cell lines by lipofectamine and electroporation. A. A comparison of chemical and physical technique efficiencies for gene transformation confirmed a higher rate of transfection for electroporation in individual cell lines. The graph shows the averages of three independent experiments. Error bars represent the standard deviation. **; P<0.01 and B. Flow cytometric analysis of EGFP expression in three independent experiments. After 48 hours of gene delivery, we analyzed transient expression of EGFP by flow cytometry. hFF cells showed the highest percentage (27%) of expression when compared with the other cells. Interestingly, a comparison of the two different human embryonic stem cells (hESCs) demonstrated that Royan H5 exhibited greater transformation potential (approximately 17 vs. 10%).

Article Snippet: In brief, 600 μl of the previously singled cells that contained 10-60 μg linearized plasmid was transferred into the electroporation cuvette (BioRad, USA, #165-2088).

Techniques: In Vitro, Transfection, Electroporation, Comparison, Transformation Assay, Standard Deviation, Expressing, Flow Cytometry

Journal: Cell Journal (Yakhteh)

Article Title: Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

doi:

Figure Lengend Snippet: Transient expression of EGFP in Royan H6 and human foreskin fibroblast (hFF) cell lines. A, D. Bright-field images of human embryonic stem cells (hESCs) and hFF cells, B, E. Fluorescent images and C, F. Merged bright-field and fluorescent images. The pictures showed that hFF responded to electroporation more efficiently than hESCs.

Article Snippet: In brief, 600 μl of the previously singled cells that contained 10-60 μg linearized plasmid was transferred into the electroporation cuvette (BioRad, USA, #165-2088).

Techniques: Expressing, Electroporation