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'''''Originally pu..."
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{{Header:Invited_article|Imaging of BacMam Transfected U-2 OS Cells |Paul Held, Peter Banks, [http://www.biotek.com/ BioTek Instruments, Inc.]}}
'''''Originally published as a '''''[http://www.biotek.com/resources/articles/imaging-bacmam-transfected-u-2-os-cells.html '''''white paper'''''] '''''by '''''[http://www.biotek.com/ '''''BioTek Instruments, Inc.''''']''''', April 15, 2013. Reprinted with permission.'''''
''Optimization of Transfection Conditions Using the Cytation™3 Cell Imaging Multi- Mode Reader and Gen5™ Data Analysis Software''
== Abstract ==
Transient transfection of tissue culture cells is a routine method to introduce novel genetic elements into cultured cells. While there are numerous methods to introduce foreign DNA into tissue culture cells the efficacy of the experimental results is dependent on adequate expression of the introduce gene. Here we describe the use of the Cytation™ 3 Cell Imaging Multi-Mode Reader to optimize and assess gene expression and transfection efficiency in live cells after transfection using a BacMam gene delivery system.
== Introduction ==
It is almost 20 years since Roger Tsien’s pioneering work with green fluorescent protein (GFP) where he showed that the GFP chromophore is formed in a chemical reaction which requires oxygen but without the need for any protein co-factor. Over the years, Tsien and collaborators constructed various GFP mutants by mutagenesis that possessed greater fluorescence quantum yield and had different excitation and emission spectra, such that a rainbow of GFP-like proteins are now available. Furthermore, the relatively easy genetic encodability of GFP and its variants as a fusion protein allows them to be used to localize proteins as they move around the cell, as well as monitor protein translation. GFP and its many colored variants are now well established tools used in cell biology, particularly with imaging techniques. For his role in the development of GFP as a preeminent tool in the cell biologist’s toolbox, Tsien was awarded the Nobel Prize in Chemistry in 2008.
There are many ways that GFP fusion proteins can be generated in live cells. A particularly useful method for gene transfer into mammalian cells involves using baculovirus, an insect cell virus that can be modified to express proteins in mammalian cells. This is commonly called BacMam (Baculovirus – Mamalian cells) – see Figure 1.
BacMam gene delivery technology is a transient expression system, which has many attributes that make it more attractive than stable expression. These attributes include rapid gene delivery, simple workflows that allow for the use of automation, relative non-toxicity compared to other methods, broadly applicable to many cell types including hard to transfect cells such as primary and stem cells and has the capacity for large foreign gene inserts up to 38 kb. It has been used over diverse applications including the protein expression of biologics, high throughput screening and very commonly for fluorescence microscopy involving the transfer of GFP fusion proteins.
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| align="center" | [[Image:BioTek-BacmamTransfectedU2OS-Fig1.jpg|450px]]
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| align="center" | '''Figure 1.''' BacMam virus.
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In this application note, we will demonstrate the utility of a novel cell imaging multi-mode reader for BacMam GFP fusion protein transfection assay development in microplates. The reader performs PMT-based whole well epi fluorescence intensity measurements from the top of microplates; and CCD-based inverted digital microscopy from the bottom of microplates. Used together, the two detection modes facilitate the optimization of BacMam GFP fusion protein transfection conditions for fluorescence microscopy applications in microplates.
== Materials and Methods ==
=== Materials ===
U-2 OS cells (Sigma Aldrich) were grown in Advanced DMEM (Cat. No. 12491) from Life Technologies supplemented with 10% FBS, 2 mM glutamine. Black sided, clear bottom 96-well (Cat. No. 3603) microplates were from Corning. TopSeal-A (Cat. No. 6050195) adhesive plate sealers were from PerkinElmer. BacMam Histone H3 reagent (Cat. No. A12894), and phenol red free DMEM/F12 media (Cat. No. 11039021) were purchased from Life Technologies.
=== Cell Culture ===
U-2 OS cells were grown in Advanced DMEM supplemented with 10% FBS, 2mM glutamine, Penicillin/ streptomycin Cell cultures were maintained at 37°C, 5% CO2 in a humidified incubator. Cultures were routinely trypsinized (0.05% Trypsin-EDTA) at 80% confluency. For experiments, cells were resuspended in phenol red free DMEM/F12 media supplemented with 10% FBS, 2 mM glutamine, and plated at 25,000 cells per well (75 μL) using the peripump dispenser of the MultiFlo™ Dispenser (BioTek Instruments).
=== BacMam Virus Titration ===
A series of 2x BacMam Histone H3 reagent solutions were prepared. BacMam virus reagent was serially diluted using phenol red free DMEM/F12 media, supplemented with 10% FBS, 2 mM glutamine. After serial dilution 75 μL aliquots of each dilution were added manually to 96-well plates containing previously plated U2OS cells in 75 μL.
=== Live Cell Staining ===
Cells nuclei were stained with Hoechst 33342 (bisBenzimide) solution (Sigma Aldrich Cat. No. 14533). A stock solution (35 mM) Hoechst 33342 solution in DMSO was diluted in phenol red free DMEM/F12 media to 10 μM to make 2x working solution immediately before use. To each well of the microplate 150 μL of the working solution was added and the plate incubated at 37°C 5% CO2 at a final concentration of 5 μM for 15 minutes. After staining plates were sealed using a clear adhesive sealer.
=== Cytation3: Imaging and Whole Well Fluorescence Measurements ===
Cytation3 is a cell imaging multi-mode microplate reader. In this work, twin dual grating monochromators provided top-reading whole well fluorescence intensity measurements; automated CCD-based digital inverted fluorescence microscopy was performed from the bottom of microplates. Microscopy light cubes were configured for DAPI, GFP and Texas Red fluorescence. The DAPI light cube is configured with a 377/50 excitation filter and a 447/60 emission filter; the GFP light cube uses a 469/35 excitation filter and a 525/39 emission filter; while the Texas Red light cube uses a 586/15 excitation and 647/57 emission filters. Both 4x and 20x microscope objectives were used in imaging studies. Exposure settings were automatically determined for each color independently on a defined control well, while focus was provided automatically on each well using the DAPI signal. Reader was controlled and data captured and analyzed using Gen5™ Data Analysis Software.
The time course of BacMam transfection was performed with the microplate remaining in the Cytation3. Atmospheric control over the microplate was maintained using the BioTek Gas Controller module such that CO2 levels were maintained at 5%. The reader also provided temperature control at 37°C ±0.5°C.
== Results ==
=== Time Course of BacMam Transfection ===
U-2 OS cells infected with BacMam Histone H3 virus will express chimeric Histone H3-GFP fusion protein in their nuclei. The chimeric protein can then be monitored via fluorescence. Infected cultures were monitored kinetically over a period of 24 hours. As demonstrated in Figure 2, distinct cellular expression of GFP fluorescence can be observed as soon as 6 hours reaching a maximum at 16-20 hours and then beginning to subside at 24 hours.