The electroporation conditions for PBMC (peripheral blood mononuclear cells), purified unstimulated T cells or NK cells are similar. Unstimulated T cells have a small cell diameter of only 7-8um and stimulated T cells grow to 10-12um diameter. Because of the small size, PBMC or unstimulated T cells require a much higher electroporaiton voltage than cell lines or stimulated T cells. The voltage is typically 1200V-1400V and only 20ul and 100ul tubes can be used under PBMC mode.

     PBMCs can also be electroporated in the Legacy mode as we used in the previous Model LE/EX machines. Under Legacy mode, PBMCs need to be cooled down to 4oC by inserting the electroporation tube completely into ice in an upward direction for 4-6 minutes. After cooling, the cells can be electroporated immediately without wiping off the water drops on the tube. The parameter is 800V-860V 20ms. Model EX+ machine can process 600ul, or up to 200M PBMCs in Legacy mode. The cooling time for 600ul tube is 7-8 minutes and the parameter is 1350-1400V 20ms.

     Fresh PBMC cells survive quite well after electroporation. The true survival rate is over 60% calculated by the ratio of survived cells/initial cells. Frozen-thawed PBMC cells need about 30-40V more voltage than fresh PBMC cells.

     Cell density affects electroporation voltage. For example, increasing from 2M cells to 4M cells in 20ul electroporation will require a voltage increase of about 20V. Donor diference also affects electroporation voltage. There might be +/- 20V difference for 20ul tubes due to donor difference. 

     Transfection efficiency is also heavily influenced by plasmid quality. Good quality GFP plasmid can produce over 80% transfection efficiency. mRNA typically has higher electroporation efficiency and for PBMC, mRNA transfection efficiency is usually >92% with good viability.2