Benefits of Transduced fibroblasts acquired on Celigo image cytometer
- Rapid, whole-well analysis of transfected/transduced cultures (1536-well to 6-well)
- Quickly identify advanced parameters for high-efficiency transfection & transduction
- Determine transient and stable transfection/transduction rates using live imaging
- Reliable analysis of many different adherent and non-adherent cell types
Introduction
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 image cytometer, these parameters can be rapidly improved by repeated imaging of one set of wells or flasks over a period of time. Reliable whole-well brightfield cell counting generates cell-based data normalized to absolute cell numbers.
GFP transfection optimization in 96-well plates without trypsinization
Fluorescent proteins
Whole-well view GFP/RFP transfected HeLa cells in a 96-well plate.
- Identifies cells in brightfield image
- Measures fluorescent protein signals in green and red channels
- Label-free, non-invasive – no need to trypsinize adherence cells
- Quantifies fluorescent protein signals on a cell-by-cell basis repeatedly on the same plate providing temporal data
- Add propidium iodide for viability of GFP transfect cells in the same well
Imaged and counted cells
(A) Cell image overlay: brightfield and green, (B) Celigo image processing software identifies all the cells using brightfield image, indicated by the red outline. Within this red outline, green fluorescent intensity was measured to gate cell populations and produce transfection efficiency.
Monitoring transfection rates and viability over 5 days
HeLa cells were transfected with a range of concentrations of a plasmid-encoding turbo-GFP1 and seeded out into a 96-well plate. To monitor cell death, propidium iodide was added to the wells in some experiments. The same plate was imaged daily over a 5-day period.
A single 96-well plate was used to optimize transient transfection of adherent HeLa cells with multiple time points.
Transduced fibroblasts acquired on Celigo image cytometer
Live whole-Well transduced fibroblasts
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 image cytometer
Live Images: Segmentation of Transduced Fibroblasts
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 image cytometer
Images and analysis of Transduced Human Foreskin Fibroblasts
Images and image analysis of human foreskin fibroblasts transduced with varying amounts of a GFP lentivirus acquired and analyzed on the Celigo cytometer.
For research use only. Not for use in diagnostic procedures.