Transfection Efficiency
Transfection Efficiency by Flow Cytometry (GFP)
Quantitative percentage of transfected cells and GFP intensity per cell.
Purpose
To quantitatively determine the percentage of transfected cells and the intensity of GFP expression at the single-cell level.
Principle
Flow cytometry analyses thousands of cells rapidly, providing precise data on transfection efficiency and expression levels.
Standard protocol
- 1
Harvesting cells
- Wash cells with PBS.
- Detach with trypsin (adherent cells) or centrifuge (suspension).
- Resuspend in PBS + 1–2% FBS (to prevent clumping).
- 2
Filtering: pass the cell suspension through a 40–70 μm filter.
- 3
Flow cytometry
- Run samples on the flow cytometer.
- Set up compensation using single-colour controls.
- Collect 10,000–20,000 events per sample.
- 4
Data analysis
- Use software (e.g., FlowJo, BD FACSDiva).
- Gate live cells (FSC/SSC).
- Set the GFP-positive threshold using the untransfected control.
- Report % GFP-positive cells and mean fluorescence intensity (MFI).
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