Quantitative Measurement of GFP Transfection
Table of Contents
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 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 20µl 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.
1. Pipette 20 µl of cell sample into a disposable counting slide.
2. Insert slide into the instrument
3. Select assay from a drop down menu
4. Click count, acquire image and view cell count, concentration, diameter and percent of GFP positive cells
|BR1: 1058||1.52x10^6 cells/mL||15.2 micron|
|GFP: 594||8.56x10^5 cells/mL||16.8 micron|
|GFP Positive 56.4%|
|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: 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|
|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|
|Cell Population||% of gated cells||Concentration
(x 10^6 Cells/ml)
|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 4 Flow 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.
|Optics Module||Fluorophores||Nucleic Acid Stains||Fluorescent Proteins|
Ex: 375 nm
Em: 450 nm
Ex: 475 nm
Em: 535 nm
|AO (acridine orange, +DNA)
Ex: 525 nm
Em: 595 nm
AlexaFluor® 555, Cy3®
|PI (propidium iodide)
EB (ethidium bromide)
Ex: 540 nm
Em: 660 nm
|PI (propidium iodide)
EB (ethidium bromide)
AO (acridine orange, +RNA)
Ex: 630 nm
Em: 695 nm
|AlexaFluor® 647, Cy5®
*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 Yeast (measured by Vision 10X)
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!
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