Journal: Scientific Reports
Article Title: Miniaturized scalable arrayed CRISPR screening in primary cells enables discovery at the single donor resolution
doi: 10.1038/s41598-025-13532-z
Figure Lengend Snippet: Comparative Evaluation of Electroporation Platforms for mRNA Delivery into Primary Human Myoblasts and T Cells. ( A ) EGFP mRNA transfection in cell dilutions of primary human skeletal muscle myoblasts using the Lonza Nucleofector. (Left) Representative fluorescent images of transfected primary human myoblasts expressing GFP at various cell dilutions. Scale bar = 200 μm. (Right) GFP expression over 48 h in primary human myoblasts in various cell dilutions ( N = 1–2). Mean values and SD are presented. ( B ) EGFP mRNA transfection in cell dilutions of primary human T cells using the Lonza Nucleofector. (Left) Representative fluorescent images of transfected primary human T cells expressing GFP at various cell dilutions. Scale bar = 200 μm. (Right) GFP expression over 48 h in primary human T cells in various cell dilutions ( N = 1–4). Mean values and SD are presented. ( C ) (Left) Schematic of the DropGenie microfluidic cartridge. (Right) Workflow schematic for using the DropGenie Transfection System. First, guides are deposited onto the substrate and cells and payload are loaded onto the DropGenie cartridge. The DropGenie Transfection System is run using user defined parameters and cells are offloaded into a plate to allow for further culture and recovery. Post-transfection, downstream assays can be conducted to confirm efficiency of transfection, such as flow cytometry, sequencing, or fluorescence microscopy. ( D ) EGFP mRNA transfection in primary myoblasts using the DropGenie Transfection System with initial cell input of 3,000 cells/edit. (Top) Confluency of primary human myoblasts over 108 h post-transfection, with an initial cell input of 3,000 cells/edit ( N = 5–12). Mean values and SD are presented. (Middle) GFP expression over 48 h of primary human myoblasts post-transfection ( N = 5–12). Mean values and SD are presented. (Bottom) Representative fluorescent image of transfected primary human myoblasts expressing GFP 48 h post-transfection at 500 V, 3 ms, 3 pulses. Scale bar = 200 μm. ( E ) EGFP mRNA transfection in primary human T cells using the DropGenie Transfection System with initial cell input of 10,000 cells/edit. (Left, Top) Confluence of primary T cells over 156 h post-transfection ( N = 8–10). Mean values and SD are presented. (Left, Middle) GFP expression over 48 h of primary human T cells post-transfection ( N = 8–10). Mean values and SD are presented. (Left, Bottom) Representative fluorescent image of transfected primary human T cells expressing GFP 32 h post-transfection at 500 V, 3 ms, 2 pulses. Scale bar = 400 μm. (Right) Quantification of GFP% positivity via flow cytometry in primary human T cells 24-hours post-transfection at 500 V, 2 ms, 2 pulses in the (Top) CD4 + and (Bottom) CD8 + T cell subsets. ( N = 4–15). Statistics were calculated using One-way ANOVA with multiple comparisons. Statistical significance is defined as ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Mean values and SD are presented.
Article Snippet: Post-transfection, cells were offloaded into Skeletal Muscle Cell Growth Media in a 96-well flat-bottom plate and imaged on the Incucyte Live-Cell Analysis System (Sartorius) at 37 °C, 5% CO 2 .
Techniques: Electroporation, Transfection, Expressing, Flow Cytometry, Sequencing, Fluorescence, Microscopy
