Introduction
Jurkat cells are an immortalized line of T lymphocyte cells that are used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptors susceptible to viral entry, particularly HIV. The Jurkat cell line (originally called JM) was established in the late 1970s from the peripheral blood of a 14 year old boy with T cell leukemia.

Lymphocyte-derived cells are known to be difficult to transfect with non-viral vectors. Most synthetic cationic reagent of the new generation transfections reagents have proven to be unsatisfactory in the transfection of T-lymphocytes. We tried our SAINT-18 transfection reagent, which can be used for a wide variety of mammalian cells, including poor transfectable cells such as primary cells. After optimization of the transfection parameters, we developed a transient transfection protocol for activated Jurkat cells. Using this protocol we consistently achieve thirty percent expression of GFP in Jurkat cells using SAINT-18 Reagent.

T-cell activation is normally triggered by the interaction of a cell surface receptor to its specific ligand molecule. This binding event triggers the rapid hydrolysis of inositol phospholipids to diacylglycerol and inositolphosphates by phospholipase C (PLC). Phorbol 12-myristate13-acetate (PMA), which has a structure analogous to the allosteric activator diacylglycerol, can also activate protein kinase C (PKC), which trigger Ca++release and mobilization, resulting in a cascade of additional cellular responses mediating T-cell activation. One of these cellular responses is the production and secretion of interleukin-2 (IL-2). Because of this ability to activate and stimulate Jurkat cells these cells are very useful in science.

Method
Jurkat T cells were cultivated in RPMI 1640 with L-glutamine and 25 mM HEPES buffer. The cell culture medium was supplemented with 10% fetal bovine serum (FBS), 0.5mM L-Glutamine, 1mM sodium pyruvate, 60µg/mL gentamycin (all Lonza) and 50 µM beta-mercaptoethanol. Cells were maintained at 37°C in a 5% CO2 humidified atmosphere.

On the day before transfection, cells were splitted in fresh medium. pCMV-eGFP plasmid DNA was isolated by midiprep (Marcherey-Nagel) following the protocol of the manufacturer. Transfection experiments were conducted using SAINT-18 transfection reagent from Synvolux Therapeutics. The SAINT-18/pDNA complex was prepared on the day of transfection in sterile polystyrene tubes (Greiner).

For a typical DNA stock of 0.5 to 2 µg/µl, 5 µg of pCMV-eGFP reporter plasmid were diluted with sterile distilled water to a concentration of 100ng/µl. For a triplicate assay 100ng PMA (phorbol 12-myristate 13-acetate, Sigma) was diluted in 133µl water and mixed with 7.5µl of the pDNA solution (750ng) and incubated for 5 minutes at room temperature. Then 10µL SAINT-18 per tube was added to form complexes and after 1-2 minutes 3x50µL of the complex was pipetted to 3 wells of a 24-wells plate. As a negative control and the determination of auto fluorescence 3µL SAINT-18 was pipetted in an empty well.

The exponentially growing jurkat cells were counted, centrifuged at 350xg for 10 minutes, and resuspended at a density of 4 x 105 per ml in pre-warmed serum-free medium (20-37°C).

250µL of this cell suspension was pipetted to each complex in the wells and incubated at room temperature for 5-15 minutes before adding 500µl complete medium (with 10% serum). The cells were further incubated at 37°C in a 5% CO2 humidified atmosphere. Avoid storing the cell suspensions longer than 15 min in serum-free medium as this reduces cell viability and gene transfer efficiency. The cells were incubated with the transfection mixture for 48 hours. FACS analysis revealed about 30% of the Jurkat cells were expressing the green fluorescent protein.

Results and Discussion

Fig. 1 Efficiency and viability were analyzed 48 hours post transfection by flow cytometric analysis (Guava Technologies). Percent eGFP positive cells after transfection with plasmid DNA (pCMV-eGFP) in combination with the stimulant PMA and the reagent SAINT-18.

For cells such as Jurkat T cells, which are notoriously difficult to transfect, a simple procedure of one transfection with SAINT-18 in the presence of the activator PMA shows a high level of the reporter gene expression. In this respect, the SAINT-18 transfection reagent surpasses other commercially available transfection reagents. Other advantages of using SAINT-18 for Jurkat transfections include good reproducibility. Furthermore, this technique allows reduced cell manipulations compared to other techniques (e.g. DEAE-Dextran, electroporation, calcium phosphate procedures), which generally need medium removal or refreshing or extra cell washing procedures. These parameters are certainly not negligible and are particularly time-consuming. In conclusion, the use of SAINT-18 as a transfection reagent for T-lymphocytes is a considerable improvement over other currently available technologies.

Protocol

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