Quantitative imaging of green fluorescent protein in cultured cells. The green fluorescent protein was originally isolated from the jellyfish and is a specialized protein that emits fluorescence when exposed to excitation light. The fcm apparatus is based on a zeiss axiovert 35 microscope with a capochromat 403 1. Lentibrite gfplc3 lentiviral biosensor sigmaaldrich. Optimization of transfection of green fluorescent protein. Our aim was to test the efficiency of ep and lipofection protocols in endometrial and testicular tissue fragments, using a naked plasmid dna encoding green fluorescent protein gfp. Transfection of pluripotent stem cells with lipofectamine. Through this protein, it has become easy to not only observe proteins as they are being made, but also observe any movements. Cells were visualized by fluorescence microscopy and processed using flow cytometry to determine transfection efficiency. Transfection of chok1 cells with expression vectors encoding gfp from two different sources, and optimization of transfection. Gfp green fluorescent protein is an extremely stable protein of 238 amino acids produced by the jellyfish aequorea victoria 1 that fluoresces in the lower green region of the visible spectrum. Metafectene pro was found to be useful to introduce expression vectors into hek293t cells as comparable to other commercially available transfection. Flim can separate probes of the same color but different fluorescence lifetimes fl.
Gfp is commonly used for monitoring gene expression, protein trafficking within intact cells and pursuing the transfected cells. These were then packaged in transfection reagent and spotted robotically onto a glass slide to form a reverse transfection array. Emd millipore s lentibrite histone h2bgfp lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of chromosomal dynamics in difficulttotransfect cell types. Transfection of leishmania tropica with green fluorescent. Chou department of chemistry, university of british columbia, vancouver, british columbia, canada abstract we report applications of twophoton excitation. Ive stably transfected hek293 cells using hd fugene. Transfection of chok1 cells with expression vectors encoding gfp from two different sources, and optimization of transfection conditions for both plasmids were performed. Fluorescence microscopy is a major tool with which to monitor cell physiology. The lentibrite lentiviral transfection yields higher efficiency and more uniform expression of gfp tubulin versus chemical transfection. Transfection of leishmania tropica with green fluorescent protein gfp gene and investigation of the in vitro drug effect article pdf available in mikrobiyoloji bulteni 532.
Techniques vary widely and include lipidbased transfection and physical methods such as electroporation. Cells are put on slides using cytospin centrifugation for fluorescence microscopy. Observe pscs transfected with a gfp reporter construct at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry, and analyze doublestranded break formation using the invitrogen geneart genomic cleavage detection kit or similar assay figure 3. Transfection efficiency analysis at 48 hr following transfection of a gfp reporter construct, cells were evaluated via microscopy and flow cytometry. Hey guys, im confused and therefore write here with the hope that someone might provide useful input. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. Accordingly, the fluorescence anisotropy dynamics of two fusion proteins tk27gfp and tk366gfp was recorded in the confocal mode by ultrasensitive. Subdiffractionlimit twophoton fluorescence microscopy. Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomerdimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase tk fused to green fluorescent protein gfp.
Tips and tricks adding 50 ng of gfp mrna to the transfection. One way to control for background levels of puncta is to determine fluorescence from untagged gfp. We also hope the manual will serve as a resource for anyone interested in the. Green fluorescent protein gfp thermo fisher scientific. Correlated fluorescence microscopy and cryoelectron. In particular, the anisotropy measured from a rigid tagging of gfp was used to monitor the orientation of septins in yeast cells 15,16. To determine the application limits of green fluorescent protein gfp as a reporter.
The percentage of cells that exhibit the reporter gives the transfection. Then, the intensity of green fluorescent protein gfp was quantified using fluorescence reader at emission wavelength 506 nm and excitation wavelength 500 nm. My transfection efficiency was 100% and then after if when i check the cells under conforcal it seems to be like 50% transfection efficiency. Green fluorescent protein gfp thermo fisher scientific us. Mapping the orientation of nuclear pore proteins in living. Because the transfection efficiency depends upon several factors, we tried to optimize the transfection. Reporter genes, such as green fluorescent protein gfp, luciferase, or. Fluorescent labeling of the nuclear envelope by localizing. To assess whether the expression of apexgbp affected the distribution of the gfptagged poi we routinely performed confocal microscopy of all gfptagged proteins apexgbp. Polarized fluorescence microscopy has previously been used to investigate macroscopically ordered systems of different geometries 1217. Qualitative analysis showed that all novel expression vectors were able to express gfp. A few cells are definitely transfected strong gfp fluorescence. Current methods manual observation using fluorescent microscope. For optimal fluorescent visualization, it is recommended to analyze the target expression level within 2448 hrs after transfection infection for optimal live cell analysis, as fluorescent.
An enhanced green fluorscent protein allows sensitive detection. Rapid characterization of green fluorescent protein fusion. We have recently used polarized fluorescence microscopy. Monitoring transfection efficiency by green fluorescent protein gfp. Fixation of samples with cells producing fluorescent.
Tips and tricks the amount of lipofectamine stem reagent required for optimal transfection depends on the amount of pscs plated and the amount of dna used. An introduction concepts and tips for better fixed sample imaging results. The frequency of both transfected and nontranfected cells were determined. The authors were able to detect fluorescence and later observe the sections by electron microscopy using a 10 nm goldlabeled secondary antibody. The specimens are incubated and monitored by livecell fluorescence microscopy for a specified period of time post infection or transfection, and are then plungefrozen in liquid ethane steps 1627. This is followed by infection or transfection of the cultured cells with fluorescently tagged viruses or viral cdna, respectively step 15.
Green fluorescent protein as indicator of nonviral. Maybe its too obvious and i just happen to miss the forest for the trees. The green fluorescent protein gfp is a protein composed of 238 amino acid residues 26. In general, the light emission of rfp has a better tissuepenetration than that of gfp and thus better imaging choice. Pdf transient transfection of cryptosporidium parvum.
Can i use fluorescence microscopy to assess the transfection efficiency of the suresilencing shrna plasmids with gfp in my model cell line of interest. Biotek white papers, 28jul15, sample preparation for fluorescence microscopy. Youve just watched joves introduction to fluorescence microscopy. Green fluorescent protein significance, benefits and. Effect of transient expression of fluorescent protein probes. Using the lunafl dual fluorescence cell counter for. Bright gfp with subnanosecond fluorescence lifetime. In that case, gfp fluorescence survived fixation in 4% paraformaldehyde with 0. Oct 22, 2012 we chose 3 fluorescent proteins of different colors, viz. Lentiviral biosensors life science research milliporesigma. High resolution, fluorescence deconvolution microscopy and. Transfection of mammalian cellsfor 293 ebna and nih3t3 cells, 14. Cell lines like cho has commonly be used in transfection experiments since they take up dna and express recombinant proteins very efficiently 4. Yet when using fluoresence microscopy only the colonies transfected with the goiegfpvector show gfp fluorescence, while none of the egfptransfected hek293 cells seem to exhibit gfp fluorescence.
Some organisms have evolved fluorescent molecules perhaps used to attract prey that, like the toxins listed above, have been harnessed for. Over the past few years gfp has become one of the most widely used tools in molecular and cell biology as the gfp gene can be expressed in non. Once i transfected them with a egfponlyvector, once i used a vector with my gene of. It is a lipid labeled with a fluorescence tag rhodamine which can be detected by fluorescence microscopy. Can i use fluorescence microscopy to assess the transfection. Measurement of cytosolic, mitochondrial, and golgi ph in. Gfp fluorescence peak fraction analysis based nanothermometer.
Visualize your crispr experiment with mission cas9gfp. Observe pscs transfected with a gfp reporter construct at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry for endpoint analysis figure 1. Microscopy allows direct visualization of transfected cells. We offer a series of gfp invitrogen celllight fusion constructs of signal. In this report, we present a modified method for fret quantification in cultured cells using conventional fluorescence microscopy. Reliable and global measurement of fluorescence resonance. The use of gfp tagging in fluorescence micros copy including fluorescence resonance energy transfer fret prior to gfp tagging, the fluorescent molecules used in fluorescence microscopy. Miyan, manchester university institute of science and technology. Improved fluorescence and dual color detection with enhanced. Cardiomyocytes were cultured on laminincoated glass bottom dishes in standard medium and transduced with lentiviral particles at a multiplicity of infection of 50. Fluorescence lifetime imaging microscopy flim measures fluorescence decay rate at every pixel of an image. A fluorescence microscope image of yeast cells expressing gfp in an autofluorescent medium a is corrected with software to remove autofluorescence to produce a clearer picture of the cells b.
The gfp rab constructs described above were expressed in cells together with various organelle markers for morphologic evaluation of their effects by immunofluorescence microscopy. Plasmids encoding the indicated proteins were transfected into hela cells 16 hours prior to imaging by wide field deconvolution fluorescence microscopy. Here, we report that certain transfection reagents can. An advanced technique known as fluorescence recovery after photobleaching, or frap, is performed by intentionally photobleaching a small region of a sample in order to monitor the diffusion rate of fluorescently labeled molecules back into the photobleached region. A fluorescence microscope image of yeast cells expressing gfp. Lentibrite rfp control lentiviral biosensor sigmaaldrich. In our hands, this has not been an issue and we routinely use confocal fluorescence microscopy to monitor the distribution of the gfp tagged protein of interest with and without the apex. Green fluorescent protein gfp is a versatile biological marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression in vivo. Within the cells, green fluorescence was analyzed via facs at 24h, 48h, and 72h following transfection.
Faq id 2894 whenever possible, use flow cytometry to determine the transfection efficiency of the suresilencing shrna plasmids encoding gfp. Transfection of human embryonic kidney cellline 293t. Celigo demonstration experiment gfp transfection efficiency measurement 2 1001403 rev a experiment. Transfection is the process by which nucleic acids are introduced into eukaryotic cells. Introduction to fluorescent proteins microscopyu enus. Apr 03, 2018 the authors were able to detect fluorescence and later observe the sections by electron microscopy using a 10 nm goldlabeled secondary antibody. Gfp transfection efficiency measurement purpose to analyze transfection efficiency over a 4 day period when treated with various amounts of a transfection compound. Fluorescence resonance energy transfer of gfp and yfp by. The green fluorescent protein gfp and its spectral variants yellow, yfp. Knight and nicholas billinton logical processes, monitoring subcellular. Finally, confirming localization of your protein of interest can be done by microscopy.
Its primary importance for current research lies in the ability of the purified jellyfish gfp gene to express the fluorescent protein in other living organisms. A dedicated filter combination for quantitative imaging of gfp should use a. To reliably measure fret, three positive control constructs in which a cyan. Localization, trafficking, and temperaturedependence of the. To assess transfection efficiency, cells were first visualized via fluorescence microscopy. B green fluorescent protein image after transfection by fluorescence microscopy. Fluorescence lifetime imaging microscopy by tcspc tdflim. Monitoring sphingolipid trafficking in cells using. Others show only quite a weak fluorescence, so i ask myself, if this is just autofluorescence or if these cells are really. Yet an arsenal of photonic techniques is available to tackle the problem and to allow effective discrimination of gfp from autofluorescence. Feb 26, 2019 the use of gfp tagging in fluorescence microscopy including fluorescence resonance energy transfer fret prior to gfp tagging, the fluorescent molecules used in fluorescence microscopy were often. C western blot analysis of sirt3 protein expression levels after transfection. Gfp can easily be visualized by standard fluorescence microscopy to track realtime subcellular localization of a protein of interest. Although green fluorescent protein can generally be imaged with a standard fluorescein filter combination if the intensity is high enough, the use of specialized filters see figure 3 will enable finetuning of signal collection to include or exclude other components.
However, measurements of organelle ph in living cells have been scarce. Apr 30, 2014 using the lunafl dual fluorescence cell counter for quantification of gfp transfection efficiency. Green fluorescence protein gfp based fluorescence resonance energy transfer fret is increasingly used in investigation of inter and intramolecular interactions in living cells. Green fluorescent protein gfp chimeras have been invaluable in cell biology for the localization of molecules in both live and fixed cells without requiring specific antibody reagents. Controls are needed to verify that the transfection protocol does not artificially induce gfplc3 puncta or cause lc3 aggregation. Identification of transfection efficiency using qualitative. How to calculate transfection efficiency the number of cells that exhibit the reporter and the number of cells that do not exhibit the reporter can be counted under a microscope or by flow cytometry. Establishment of green fluorescent protein expressing cho. Transfection of gfp green fluorescent protein into cho cells. Gfp fusion protein localized to the plasma membrane was characterized in detail 12. Transfection protocol lipofectamine 3000 transfection reagent. Confocal microscopy green fluorescent protein olympus. Transient transfection of cryptosporidium parvum using green fluorescent protein gfp as a marker article pdf available in molecular and biochemical parasitology 1682. The discovery of green fluorescent protein in the early 1960s ultimately heralded a new era in cell biology by enabling investigators to apply molecular cloning methods, fusing the fluorophore moiety to a wide variety of protein and enzyme targets, in order to monitor cellular processes in living systems using optical microscopy.
Subdiffractionlimit twophoton fluorescence microscopy for gfp tagged cell imaging qifeng li, sherry s. A fluorescence microscope image of yeast cells expressing gfp in an autofluorescent medium a is corrected with software to remove autofluorescence to produce. High resolution, fluorescence deconvolution microscopy and tagging with the auto. To evaluate the transfection efficiency, we expressed enhanced green fluorescent protein egfp and analyzed by fluorescent microscopy. Our results demonstrate we can localize proteins at highresolution with cotransfection of a gfptagged protein of interest and apexgbp. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. The green fluorescent protein has gained significant attention in biology, medicine and research and has been described as the microscope of the twenty first century for a very good reason.
Fluorescent imaging fli is basedon fluorescent proteins, such as rfp and gfp. Modular detection of gfplabeled proteins for rapid screening. You will need to switch back and forth between the fluorescent and brightfield modes to make this estimate. Fluorescence microscopy of live cells uses either genetically encoded fluorescent proteins e. Bioluminescent and fluorescent imaging vectors cat. What is the best fixation for fluorescence microscopy of. Gfpexpressing and nontransfected cells in the treated sample.
Transfection of chok1 cells with expression vectors encoding gfp from two different sources, and opti. Several mutants of the aequorea victoria green fluorescent protein gfp displayed a phdependent absorbance and fluorescent. Comparison of flow cytometry, fluorescence microscopy and. Gfptagging in fluorescence microscopy news medical. Huvecs were transfected with gfp tubulin plasmids using lipofectamine 2000 top row or lentibrite gfp tubulin lentiviral particles bottom row. Gfp can be excited by the 488 nm laser line and is optimally detected at 510 nm. The green fluorescent protein gfp from the jellyfish aequorea victoria has. Lentibrite histone h2bgfp lentiviral biosensor sigmaaldrich. Acgfp is a green fluorescent protein derived from aequorea coerulescens and has silent mutations that create an open reading frame almost entirely composed of preferred human codons haas et al. After 24 h, these cells were observed under confocal fluorescence microscope. To assess transfection efficiency, cells were first visualized via fluorescence microscopy for qualitative assessment of protein expression, morphology, and viability figure 1. Transfection reagents are highly efficient for dna and sirna transfection.
The spectral qualities of gfp and yfp make them one of the most efficient fret donoracceptor couples available. Gfptagging in fluorescence microscopy medical news. Modular detection of gfplabeled proteins for rapid. Hek293t cells were grown over the surface of the array until confluent and gfp fluorescence visualized by confocal microscopy. Images demonstrate the translocation of gfp lc3 from diffuse nuclearcytosolic localization to discrete cytoplasmic puncta. Green fluorescent protein an overview sciencedirect topics. Novel dual emission, phsensitive variants of the green fluorescent protein gfp have been constructed and are suitable for ratiometric emission measurements in vivo. Green fluorescent protein variants as ratiometric dual. However, the emission peaks of gfp 510 nm and yfp 527 nm are spectrally too close for separation by conventional fluorescence microscopy.
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