2 Materials
2.1 Cells and Cell
Culture
1. Mouse embryonic fibroblasts (MEFs).
2. Dulbecco’s Modified Eagle’s Medium (DMEM).
3. 10% (v/v) fetal bovine serum (FBS).
4. Nonessential amino acids.
5. Mouse cardiomyocytes.
6. H9C2 (mouse cardiomyoblasts) cells.
7. NK-92 cells.
8. RPMI 1640.
2.2 Biomolecules
Used
1. Hoechst (excitation/emitting wavelength, 350/461 nm).
2. CAR (chimeric antigen receptor) plasmids.
3. OSKM Plasmids (OCT-4, SOX2, KCL-1, and c-MYC transcription factors, OSKM encoded on a pCAG plasmid backbone) labeled with YOYO-1 dye.
4. miR-29b molecular beacon.
5. Trypsin (0.05%).
6. JC-1 dye.
2.3 Nanoelectroporation
1. 3D NEP System (Fig. 1a) [12].
2. Power supply (Gene Pulser Xcell system, Bio-Rad).
3. Wires to connect power source with the electrodes in transfection region.
4. Bottom planar electrode (gold coated stainless steel plate).
5. Top Rod Electrode (copper rod).
6. Poly(methyl methacrylate) micromilled support platform
(Fig. 1b).
7. Poly(methyl methacrylate) micromilled clamp (Fig. 1b).
8. Trizol.
3 Methods
3.1 Prepare
Nucleotides
1. OSKM plasmid (OCT-4, SOX2, KCL-1, and c-MYC transcription factors, and RFP encoded on a pCAG plasmid backbone)
is purchased from Addgene.
2. CAR plasmid is synthesized as per the instructions published in
the research work [4].
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Lingqian Chang et al.
2.1 Cells and Cell
Culture
1. Mouse embryonic fibroblasts (MEFs).
2. Dulbecco’s Modified Eagle’s Medium (DMEM).
3. 10% (v/v) fetal bovine serum (FBS).
4. Nonessential amino acids.
5. Mouse cardiomyocytes.
6. H9C2 (mouse cardiomyoblasts) cells.
7. NK-92 cells.
8. RPMI 1640.
2.2 Biomolecules
Used
1. Hoechst (excitation/emitting wavelength, 350/461 nm).
2. CAR (chimeric antigen receptor) plasmids.
3. OSKM Plasmids (OCT-4, SOX2, KCL-1, and c-MYC transcription factors, OSKM encoded on a pCAG plasmid backbone) labeled with YOYO-1 dye.
4. miR-29b molecular beacon.
5. Trypsin (0.05%).
6. JC-1 dye.
2.3 Nanoelectroporation
1. 3D NEP System (Fig. 1a) [12].
2. Power supply (Gene Pulser Xcell system, Bio-Rad).
3. Wires to connect power source with the electrodes in transfection region.
4. Bottom planar electrode (gold coated stainless steel plate).
5. Top Rod Electrode (copper rod).
6. Poly(methyl methacrylate) micromilled support platform
(Fig. 1b).
7. Poly(methyl methacrylate) micromilled clamp (Fig. 1b).
8. Trizol.
3 Methods
3.1 Prepare
Nucleotides
1. OSKM plasmid (OCT-4, SOX2, KCL-1, and c-MYC transcription factors, and RFP encoded on a pCAG plasmid backbone)
is purchased from Addgene.
2. CAR plasmid is synthesized as per the instructions published in
the research work [4].
72
Lingqian Chang et al.