Nexcelom Bioscience

978-327-5340

Benefits of Transduced fibroblasts acquired on Celigo Cell Imaging Cytometer

  1. Rapid, whole-well analysis of transfected/transduced cultures (1536-well to 6-well)
  2. Quickly identify optimal parameters for high-efficiency transfection & transduction
  3. Determine transient and stable transfection/transduction rates using live imaging
  4. 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 cell imaging cytometer, these parameters can be rapidly optimized by repeated imaging of one set of wells or flasks over a period of time. Accurate whole-well bright field 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

Whole-well view GFP/RFP transfected HeLa cells in a 96-well plate.

  • Identifies cells in bright field 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

Imaged and counted cells

(A) Cell image overlay: bright field and green, (B) Celigo image processing software identifies all the cells using bright field 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

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 Cell Imaging Cytometer

Live Whole-Well Transduced Fibroblasts

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 Cell Imaging Cytometer

Live Images: Segmentation of Transduced Fibroblasts

Live Images of 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 Cell Imaging Cytometer

Images and Analysis of Transduced Human Foreskin Fibroblasts

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.

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