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GFP Transfection Efficiency Measurement

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Purpose To analyze transfection efficiency over a 4 day period when treated with various amounts of a transfection compound.
Existing Method(s) Manual observation using a fluorescent microscope
Target Cell Type 293H cells
Experiment Plan Plate transfected cells and measure transfection efficiency for 4 days
Hypothesis Higher viral dosage treatments will lead to higher GFP numbers and fluorescent intensities

Celigo Setup

Plate Type Greiner 655090 96-well black wall clear bottom
Scan Channels Bright field + Green
Resolution 1 µm/pixel
Scan Area Whole well
Analysis Method Confluence Ratio: Confluence 1 + 2
Scan Frequency Daily
Scan Time ~8 min

Assay Protocol and Plate Setup

Goal:

To analyze transfection efficiency over a 4 day period when treated with various amounts of a transfection compound.

Protocol:

Cell preparation

  • Transfected cells were collected and plated on Day 0 following the plate map below

gfp transfection efficiency plate map

  • After plating, the plate was centrifuged to settle the cells in the bottom of the dish, in order to image the cells in a monolayer for optimal focus
  • Cells were then treated with compounds following the plate map below

gfp transfection efficiency treated plate map

  • The plate was then imaged using Celigo for bright field and GFP green fluorescence
  • The plate was imaged and analyzed on Day 0, 1, 2, and 3

Data Collection

  1. After centrifuging the plate, the plate was scanned using the Celigo
  2. The scanning parameters for 2 channels were set up, where Confluence channel 1 + 2 are GFP and bright field, respectively

Data Analysis

  • The images were analyzed to measure the total area of cell coverage in bright field and in fluorescence
  • The automatically calculated confluence ratio indicates GFP transfection efficiency as a percentage of the total cell area

Results

1. GFP expression fluorescent images

  • The GFP fluorescent images showed differences between low and high compound treatment
  • The GFP expression plate view is shown below

gfp transfection efficiency plate level data

2. GFP transfection efficiency measurement results

  • Whole-well fluorescent images reveal the GFP expression level for cells treated with high and low concentration of compound

gfp transfection efficiency celigo whole-well

  • The GFP transfection percentage for 60,000 cells/well showed no expression for the negative control, low compound treatment showed 60%, and the high compound treatment showed 100%
  • The results were similar for the 80,000 cells/well

gfp transfection efficiency percentage data

3. Bright field confluence level

  • The bright field images showed that at high compound treatment, there is a reduction in cell confluency, in comparison to the low compound treatment

gfp transfection efficiency confluence

gfp transfection efficiency confluence data

  • At 60,000 cells/well, the high compound treatment showed a large decrease in cell confluency
  • At 80,000 cells/well, the high compound treatment did not show any decrease in cell confluency

4. GFP transfection time-course images

  • Green fluorescent images showed an increase in the number of GFP-positive cells over 3 days of culture
  • The low compound treatment resulted in fewer GFP-expressing cells compared to the high compound treatment

gfp transfection efficiency confluence data

Conclusion

  • The GFP transfection efficiencies were highly dependent on the compound treatment
  • At lower seeding density, the cells showed higher damage due to high compound treatment
2018-10-11T18:25:24+00:00