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Celetrix Electroporation

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Jurkat Electroporation Protocol

Jurkat cells are hard to transfect with lipid reagents. With Celetrix electroporation, Jurkat cells reach >90% transfection efficiency and keep growing fast afterwards. Below are our protocol and recommended parameters.

Jurkat Transfection Results

Side-by-side brightfield and fluorescence microscopy images demonstrating high GFP expression in Jurkat cells using Celetrix electroporation.
18 h: GFP expression in Jurkat cells
Celetrix electroporation of Jurkat cells: flow cytometry shows 90.27% GFP+ cells 18 h after transfection
Flow cytometry: 90.27% GFP+ Jurkat cells

Recommended Parameters for Jurkat

20 µl tube1–2×106 cells, 2 µg plasmid, Cell Line mode 500–540 V
100 µl tube5–10×106 cells, 10 µg plasmid, Cell Line mode 500–540 V
200 µl / 1 ml tubeCell Line mode 1200–1350 V
BufferCeletrix electroporation buffer
Recovery mediumRPMI 1640 + 10% FBS, antibiotic-free

Step-by-Step Jurkat Electroporation Protocol

  1. Culture Jurkat cells to 50–70% of their maximum density.
  2. Spin down cells at 1000–1200 rpm for 3 min.
  3. Resuspend in Celetrix electroporation buffer (1–2×106 cells per 20 µl).
  4. Add plasmid DNA, fill the tube until a small dome forms, and cap it.
  5. Electroporate right away in Cell Line mode at 500–540 V.
  6. Transfer cells to warm, antibiotic-free RPMI 1640 + 10% FBS. GFP is visible within 1–3 h and stronger overnight.

Jurkat Knockout with Cas9 RNP

Celetrix electroporation of Cas9 RNP knocks out CD3e in Jurkat cells: flow cytometry shows 92% CD3e-negative cells
Cas9 RNP knockout: CD3e+ Jurkat cells drop from 93.39% to 7.95%.

For a 20 µl Celetrix tube:

Cells1–2×106 cells, pelleted
Cas9 protein5 µg
sgRNA50–100 pmol (for multiple sgRNAs, add Cas9 protein last)
RNP assemblyMix Cas9 and sgRNA in 22 µl Celetrix electroporation buffer; incubate 10 min at room temperature
ElectroporationAdd the RNP mix to the cell pellet, mix well and electroporate (about 25 µl total)

Tips for Jurkat

  • Cas9 RNP usually needs a slightly higher voltage than plasmid.
  • About 10–20% cell death is normal; if there is a lot of cell death, lower the voltage by 20–30 V.
  • Keep cells as single cells and electroporate each tube right after loading.
  • More tips in our electroporation optimization guide.

Jurkat Electroporation FAQs

Electroporation acts at the cell membrane, so its small toxicity stays at the membrane and has little effect inside the cell. Lipid and other chemical transfection reagents affect not only the membrane but also components inside the cell. That is why Celetrix electroporation keeps cell toxicity low and Jurkat cells healthier than transfection reagents. Learn how the voltage falls on the cell membrane in our electroporation mechanism page.

On Celetrix electroporators, use Cell Line mode at 500–540 V for 20 µl and 100 µl tubes, and 1200–1350 V for 200 µl and 1 ml tubes. See all Celetrix recommended parameters.

The best voltage depends on your sample. Higher cell density needs a slightly higher voltage. With larger plasmids, cells tolerate high voltage less well, so lower the voltage slightly. That is why Celetrix gives a voltage range instead of one fixed voltage: choose a value within the range to match your cell density and plasmid size.

For Celetrix electroporation, use 1–2×106 Jurkat cells per 20 µl tube or 5–10×106 cells per 100 µl tube.

Yes. Celetrix recommends Cas9 RNP for Jurkat gene editing: 5 µg Cas9 protein with 50–100 pmol sgRNA per 20 µl tube. RNP avoids the toxicity of Cas9 plasmids.

Move cells into warm, antibiotic-free RPMI 1640 + 10% FBS right after the pulse. With Celetrix electroporation, about 10–20% cell death is normal; if there is a lot of cell death, lower the voltage by 20–30 V.

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