A solution for highly efficient electroporation of primary cytotoxic T lymphocytes.

BACKGROUND: Cytotoxic T lymphocytes (CTLs) are central players in the adaptive immune response. Their functional characterization and clinical research depend on efficient and reliable transfection. Although various methods have been utilized, electroporation remains the preferred technique for transient gene over-expression. However, the efficiency of electroporation is reduced for human and mouse primary CTLs. Lonza offers kits that effectively improve plasmid DNA transfection quality. Unfortunately, the removal of key components of the cell recovery medium considerably reduced the efficiency of their kit for CTLs. Our aim was to develop a new recovery medium to be used with Lonza's Nucleofector system that would significantly enhance transfection rates. RESULTS: We assessed the impact of different media in which the primary CTLs were placed to recover after electroporation on cell survival, transfection rate and their ability to form an immunological synapse and to perform exocytosis. We transfected the cells with pmax-GFP and large constructs encoding for either CD81-super ecliptic pHluorin or granzyme B-pHuji. The comparison of five different media for mouse and two for human CTLs demonstrated that our new recovery medium composed of Opti-MEM-GlutaMAX supplemented with HEPES, DMSO and sodium pyruvate gave the best result in cell survival (> 50%) and transfection rate (> 30 and 20% for mouse and human cells, respectively). More importantly, the functionality of CTLs was at least twice as high as with the original Lonza recovery medium. In addition, our RM significantly improved transfection efficacy of natural killer cells that are notoriously hard to electroporate. CONCLUSION: Our results show that successful transfection depends not only on the electroporation medium and pulse sequence but also on the medium applied for cell recovery. In addition, we have reduced our reliance on proprietary products by designing an effective recovery medium for both mouse and human primary CTLs and other lymphocytes that can be easily implemented by any laboratory. We expect that this recovery medium will have a significant impact on both fundamental and applied research in immunology.

Electrophysiological Characterization of eGFP-Labeled Intrastriatal Dopamine Grafts.

Substitution of degenerated dopaminergic (DA) neurons by intrastriatally transplanted ventral mesencephalon (VM)-derived progenitor cells has been shown to improve motor functions in parkinsonian patients and animal models, whereas investigations of electrophysiological properties of the grafted DA neurons have been rarely performed. Here we show electrophysiological properties of grafted VM progenitor cells at different time intervals up to 12 weeks after transplantation measured in acute brain slices using eGFP-Flag transfection to identify the graft. We were able to classify typical DA neurons according to the biphasic progression (voltage "sag") to hyperpolarizing current injections. Two types of DA-like neurons were classified. Whereas type 1 neurons were characterized by delayed action potentials after hyperpolarization and irregular spontaneous firing, type 2 neurons displayed burst firing after hyperpolarization, spontaneous bursts, and regular firing. Comparison to identified DA neurons in vitro indicates a high integration of the intrastriatally grafted neurons, since in vitro cultures displayed regular firing spontaneously, whereas grafted identified DA neurons showed irregular firing. Additionally, type 1 and type 2 neurons exhibited a slight increase in the spontaneous firing frequency over time intervals after grafting, which might reflect a progressive integration of the grafted DA neurons. Our results provide evidence of the differentiation of grafted VM progenitor cells into mature integrated DA neurons, which are shown to replace the missing DA neurons functionally early after grafting.