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Updated: Jul 18, 2026
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Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards
Published on: February 25, 2017
Quantification of EGFP expression on Molt-4 T cells using calibration standards
Y Gerena-López1, J Nolan, L Wang
1Department of Pharmacology, University of Puerto Rico, School of Medicine, San Juan, Puerto Rico 00935. ygerena@rcm.upr.edu
Summary
New calibration standards enable accurate quantification of enhanced green fluorescent protein (EGFP) expression in cells using flow cytometry. This method overcomes previous limitations, providing reliable measurements in molecules of equivalent soluble fluorochrome (MESF) units.
Area of Science:
- Cell Biology
- Biotechnology
- Molecular Biology
Background:
- Enhanced green fluorescent protein (EGFP) is crucial for assessing gene expression.
- Quantifying EGFP expression via flow cytometry has been hindered by a lack of calibration standards.
Purpose of the Study:
- To characterize EGFP calibration standards under varying environmental conditions.
- To quantify EGFP expression in transfected Molt-4 T cells using flow cytometry and these standards.
Main Methods:
- EGFP standards were tested across pH (5.0-9.0), temperature (37-80°C), and osmolality (100-600 mOsm/kg) ranges.
- Molt-4 T cells were transfected with pEGFP-N2 and EGFP expression quantified by flow cytometry using standards and the Cytofluor technique.
Main Results:
- EGFP standard fluorescence intensity increased with pH and remained stable within specific temperature and osmolality ranges.
- Transfected cells showed high EGFP expression averaging 8,098 MESF and low expression averaging 3,808 MESF.
- Flow cytometry and Cytofluor techniques yielded comparable MESF values.
Conclusions:
- Calibration standards are feasible for quantifying cellular EGFP expression.
- Monitoring environmental effects on fluorescence is vital for accurate EGFP quantification in living cells.
