Transfection & Transduction Optimization

Optimization of cellular transfection and transduction includes choosing a protocol, determining the appropriate mass of plasmid/virus, and evaluating the optimum time after transfection/transduction for the best expression of the construct of interest. Using fluorescent reporters and non-destructive in situ imaging on Celigo, these parameters can be rapidly optimized by repeated imaging of one set of wells or flasks over a period of time. Accurate whole-well brightfield cell counting generates cell-based data normalized to absolute cell numbers. Cell-based fluorescence quantification on Celigo provides several benefits:

• Rapid, whole-well analysis of transfected/transduced cultures (1536-well to 6-well)
  
• Quickly identify optimal parameters for high-efficiency transfection & transduction
  
• Determine transient and stable transfection/transduction rates using live imaging
  
• Generate time course data by repeat imaging of the same wells or flasks
  
• Evaluate antibiotic induction of expression and antibiotic selection methods
  
• Reliable analysis of many different adherent and non-adherent cell types

Transduced fibroblasts acquired on Celigo. Live whole-well (12-well plate; left) and zoomed images (right) of human foreskin fibroblasts transduced with a GFP lentivirus.

Segmentation of transduced fibroblasts on Celigo. Live images of segmentation using the Expression Analysis application. Nuclei are segmented as the mask (left) and GFP-positive nuclei are segmented as the target (right) to determine transduction efficiency.

Transduction optimization on Celigo. Images and image analysis of human foreskin fibroblasts transduced with varying amounts of a GFP lentivirus acquired and analyzed on Celigo.

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