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
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.
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.