Bio-orthogonal mediated transfection (SnapFect) evaluation and comparison. (A) Fluorescent image of native C3H10T1/2 cells with DAPI Nuclei stain. (B) Fluorescent image of native C3H10T1/2 cells treated with oxyamine containing DNA-lipoplex. No transfection occurred since the cells did not present ketone groups on their cell surface. (C) Fluorescent image of native CH310T1/2 cells and (D) image of the cells presenting ketone groups exposed to GFP oxyamine lipoplex after 24 hours. GFP expression resulted due to specific bio-orthogonal mediated transfection. (E) Representative image of RFP transfected fibroblast cells via click chemistry mediated transfection. (F) A protein gel showing the expression of RFP is mediated by the correct pairing of the bio-orthogonal chemistry functional groups on the cell surface and on the DNA-lipoplex. (O+ and K+) represent oxyamine containing lipoplexes (O+) and ketone presenting cells (K+) were used for RFP transfection. (O- and K-) represent conditions where either oxyamine or ketone was not present on the cell surface or DNA-lipoplex. Only the correct combination resulted in bio-orthogonal mediated transfection. These experiments were performed over 10 times and showed similar results. (G, H) Luciferase transfection assay for fibroblasts using the bio-orthogonal mediated transfection strategy. (H) Comparison of luciferase assay transfection for the bio-orthogonal mediated strategy (SnapFect) and leading commercial transfection reagents. The luciferase assays were replicated over 10 times and averaged (p < 0.05) (I-K) Comparison of viability, efficiency and amount of cells transfected between SnapFect and other commercial transfection reagents. Initially 5 million cells were used and the amount of surviving cells that were transfected resulted in the per cent efficiency of transfection. SnapFect has the highest ratio of viable cells to viable and transfected cells and therefore does not require further separation of cells and requires less cells for transfection. For eg. Snapfect requires only 5 million cells to generate 3.4 million transfected cells without post sorting of cells, whereas Lipofectamine and Viafect require 18 million and 11.5 million cells respectively to obtain 3.4 million transfected cells with the additional step of separating viable transfected versus viable non-transfected cells. Each of the experiments (I-K) were replicated over 30 times and averaged.