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Quantitative Measurement of GFP Transfection
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Introduction to GFP

Green Fluorescent Protein (GFP) is a 26.9 kDa protein first identified in crystal jellyfish, Aequorea victoria. It was discovered that when exposed to blue or ultraviolet light the protein fluoresces green. After GFP was first expressed in E. coli in 1994 it was soon confirmed that GFP can also be successfully expressed in other organisms as well. Since then, not only have many fluorescent proteins of different colors been generated, but their function is enhanced to provide a faster and stronger fluorescent signal.

GFP Applications

  • GFP is often used as a reporter of gene or protein expression. By detecting GFP expression it is possible to quantify the transfection/transduction efficiency.  
  • By staining the cells with propidium iodide we can monitor the viability of the culture during GFP expression.
  • In cultures that are co-transduced with GFP and RFP, the Cellometer has the capability to capture, analyze, and report the population of GFP positive, RFP positive, or dual positive.

Acquiring GFP Expression Efficiency

With the Cellometer Vision CBA, just 20 µl of sample is added to the Cellometer Counting Chamber. Imaging and analysis of GFP expression is completed in less than 60 seconds. Bright field and fluorescent cell images can be viewed to check cell morphology and verify cell counting. Total cell count, concentration, and mean diameter are automatically displayed.

Quantifying GFP Efficiency in 4 Easy Steps

1. Pipette 20 µl of sample into a disposable slide.

Pipette 20 µl of cell sample into a disposable counting slide

2. Insert slide into the instrument

Insert slide in to Cellometer

3. Select assay from a drop down menu

select gfp transfection assay

4. Click count, acquire image and view cell count, concentration, diameter and percent of GFP positive cells

GFP transfection results: cell count, concentration, and diameter

GFP Expression in 293T Cells

Automatically measure the number and percent of GFP positive cells in the population.

Bright Field and Fluorescent 293T Cell Images

GFP Expression in 293T Cells
Count Concentration Mean Diameter
BR1: 1058 1.52x10^6 cells/mL 15.2 micron
GFP: 594 8.56x10^5 cells/mL 16.8 micron
GFP Positive 56.4%

RFP Expression in RWPE-1 Cells

Automatically measure the number and percent of RFP positive cells in the population.

Bright Field and Fluorescent RWPE-1 Cell Images

RFP Expression in RWPE-1 Cells
Count Concentration Mean Diameter
BR1: 1216 1.77x10^6 cells/mL 19.6 micron
RFP: 596 8.70x10^5 cells/mL 21.2 micron
RFP Positive 49.2%

GFP Expression and Propidium Iodide Viability in Mouse Embryonic Stem Cells

Monitor cell health by adding PI to the GFP expression assay. The number and percent of the dead, PI positive cells, are shown along with the GFP expression readout.

Bright Field and Fluorescent Cell Images of Mouse Embryonic Stem Cells

GFP expression and  PI viability
Count Concentration Mean Diameter Ratios
GFP: 452 cells 1.29x10^6 cells/mL 12.2 microns GFP Positive: 17.7%
PI Positive: 46.7%
Dual Positive: 0.1%
PI: 1203 cells 3.39x10^6 cells/mL 9.4 microns
BR Total Cell: 2570 cells 7.26x10^6 cells/mL 10.0 microns

Co-Expression of GFP/RFP in T-cells

Image and Analyze double positive GFP/RPF cell populations. The Cellometer automatically reports each population as well as number and percent of dual positive cells.

Bright Field and Fluorescent T-Cell Images

Co-expression GFP/RFP Expression
Count Concentration Mean Diameter Ratios
GFP: 368 cells 5.20x10^5 cells/mL 10.3 microns GFP Positive: 38.4%
RFP Positive: 69.5%
Dual Positive: 38.2%
RFP: 667 cells 9.42x10^5 cells/mL 10.1 microns
BR Total Cell: 962 cells 1.36x10^6 cells/mL 9.0 microns

Detecting Cells Expressing Weak GFP Signal

Data Plot Gated for Fluorescent Protein Expression

GFP Weak Signal Graph

Cell Population % of gated cells Concentration
(x 10^6 Cells/ml)
Total 100.00 1.52
GFP Protein + 61.63 0.93
GFP Protein - 31.66 0.48

If the GFP signal strength is low, data acquired by the Cellometer can be exported to FCS Express Software for analysis. A histogram is generated based on GFP negative and GFP positive populations. Gating can be manually optimized directly on the histogram with automatic update to the associated data table.

Measuring GFP Positive Cell Population in Primary Mouse Bone Marrow Cells

Export and analyze Cellometer captured data in flow cytometry software FCS Express. By plotting the mean fluorescent intensity of the GFP signal we can determine the percent of GFP positive cells in the primary bone marrow sample.

Quantify GFP expression using FCS Express Software

Measuring GFP Positive Cell Population in Primary Mouse Bone Marrow Cells

Bright Field Counted Image

Bright Field Counted Image Primary Mouse Bone Marrow Cells

Fluorescence Image

Fluorescence Image Primary Mouse Bone Marrow Cells

Other Fluorescent Proteins

For Cellometer Vision CBA, customizable fluorescent optics modules are available for specific fluorescent proteins.

Fluorescent Proteins dsRed YFP CFP tdTomato Saphire

Fluorescent Optics Modules for the Detection of Fluorescent Proteins

Optics Module Fluorophores Nucleic Acid Stains Fluorescent Proteins
Ex: 375 nm
Em: 450 nm
AlexaFluor® 350 DAPI
Hoechst 33342
Hoechst 33258
Ex: 475 nm
Em: 535 nm
AlexaFluor® 488
AO (acridine orange, +DNA)
Ex: 525 nm
Em: 595 nm
AlexaFluor® 546
AlexaFluor® 555, Cy3®
PE (R-phycoerythrin)
Rhodamine B
PI (propidium iodide)
EB (ethidium bromide)
SYTOX® Orange
Ex: 540 nm
Em: 660 nm
AlexaFluor® 647
Nile Red
PI (propidium iodide)
EB (ethidium bromide)
AO (acridine orange, +RNA)
Ds Red
Ex: 630 nm
Em: 695 nm
AlexaFluor® 647, Cy5®
APC (allophycocyanin)
SYTOX® Red Crimson

*This table is a partial list of compatible fluorophores, nucleic acid stains, and fluorescent proteins. Please contact Nexcelom technical support regarding compatibility of other reagents.

Sytox, AlexaFluor, and Cy are trademarks of Life Technologies.

GFP Expression in Various Mammalian Cells

Shown here are several examples of GFP expressing cell lines. A bright field and a GFP image was collected for each cell type.

Bright Field and Fluorescent COS-7 Cell Images

GFP Expression in COS-7

Bright Field and Fluorescent 293F Cell Images

GFP Expression in 293F

Bright Field and Fluorescent HeLa Cell Images

GFP Expression in HeLa

Bright Field and Fluorescent 3T3 Cell Images

GFP Expression in 3T3

Bright Field and Fluorescent H1299 Cell Images

GFP Expression in H1299

Bright Field and Fluorescent K562 Cell Images

GFP Expression in K562

GFP Expression in Yeast (measured by Vision 10X)

GFP expression in yeast

Conclusion & Cellometer Selection

Cellometer image cytometry may be effectively utilized to determine GFP or other fluorescent protein transduction/transfection efficiency. Furthermore, it may be used to study not only GFP-expressing mammalian cell lines, but also GFP-expressing yeast cells. This robust instrument platform along with separate software-solutions provides researchers with the ability to detect strong and weak fluorescent signals.

Please contact an application specialist to determine which Cellometer is right for you!

Ask a Cellometer Specialist

Publications Using Cellometer for GFP Detection

  1. Ho YK, Zhi HJ, DeBiaso D, Philip S, Shih HM, Giam CZ. (2012) HTLV-1 Tax-Induced Rapid Senescence Is Driven by the Transcriptional Activity of NF-kappa B and Depends on Chronically Activated IKK alpha and p65/RelA.  Journal of Virology 86(17): 9474-9483
  2. Glazer ES, Zhu CH, Massey KL, Thompson CS, Kaluarachchi WD, Hamir AN, Curley SA. (2010) Noninvasive Radiofrequency Field Destruction of Pancreatic Adenocarcinoma Xenografts Treated with Targeted Gold Nanoparticles. Clinical Cancer Research 16(23): 5712-5721
  3. Chetram MA, Odero-Marah V, Hinton CV.  (2011) Loss of PTEN Permits CXCR4-Mediated Tumorigenesis through ERK1/2 in Prostate Cancer Cells. Molecular Cancer Research 9(1): 90-102
  4. Miller D, Reynolds GE, Mejia R, Stark JM, Murnane JP. (2011) Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair. DNA Repair 10(5): 536-544
  5. Kainov DE, Muller KH, Theisen LL, Anastasina M, Kaloinen M, Muller CP.  (2011) Differential Effects of NS1 Proteins of Human Pandemic H1N1/2009, Avian Highly Pathogenic H5N1, and Low Pathogenic H5N2 Influenza A Viruses on Cellular Pre-mRNA Polyadenylation and mRNA Translation. Journal of Biological Chemistry 286(9): 7239-7247