For suspension Jurkat cells, which is more suitable: electroporation or chemical transfection reagents? I'd like to hear from people who have used them.

For batch GFP screening in 96-well plates with suspension cells, I prefer chemical transfection combined with reducing the cell number per well. Many failures occur because suspension cells settle unevenly; mix before adding samples, and after adding, perform plate centrifugation at 200g for 2 min to allow more thorough contact with the complexes. Reagents to consider include Thermo Lipofectamine 3000 with P300 enhancer, or Polyplus jetOPTIMUS. I have compared them, and the latter has slightly lower toxicity to Jurkat cells. Use 0.1-0.2 μg total DNA per well in a 96-well plate. At 16-24 h after transfection, centrifuge and change the medium to increase the positive rate. Shipping time: in-stock items in Guangzhou/Pearl River Delta are generally 1-3 days; specific timing is subject to distributor inventory.

For suspension cells like Jurkat, if the sample volume is not large but there are many 96-well plate assays, I prefer electroporation, especially the Lonza 4D 96-well module or BTX 96-well, which offer good consistency in batch loading. However, the instrument platform must be considered; if the lab does not have the instrument, the one-time cost of purchasing buffers and consumables is not low. Among chemical transfection reagents, TransIT-X2 or jetOPTIMUS can be tried; they are more stable for suspension cells than regular Lipofectamine 3000. Operationally, first centrifuge at 500g and resuspend, add the DNA-reagent complex, with a total volume of 20-30 μL per well in a 96-well plate, shaking while adding. Shipping is generally 2-3 days via regional distributors.

If you already have an electroporator, do not hesitate to use electroporation; suspension Jurkat chemical methods are prone to batch-to-batch variability due to cell status. If you do not have an instrument, there is no need to buy one specifically; first try it in a nearby lab. Chemical transfection is simple to perform and can be batched: pre-seed cells in a 96-well plate with serum-containing medium, mix the reagent and DNA in Opti-MEM, incubate at room temperature for 10 minutes, and add well by well. TransIT-X2 is recommended: for 1 μg GFP plasmid, use 3 μL reagent, follow the instructions to set up a gradient, and choose the condition with both high 24 h GFP-positive rate and cell viability. Ask your local distributor about pricing and lead time; some can provide a 0.1 mL trial size.

The efficiency of suspension chemical transfection for Jurkat cells is generally not high. Although electroporation damages the cells, getting the nucleic acids in is what matters. Also waiting to hear others' experience.

Based on our comparison, electroporation achieves high efficiency but low viability, whereas chemical reagents are gentler but their efficiency varies from batch to batch.