Name: assessment of gfp expression and viability using the tali image-based cytometer
Uploaded: 2011-11-17T13:00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer at JoVE.com

1. Staining Cells with the Tali Viability Kit - Dead Cell Red Reagent
<ol>
 <li>Begin this procedure with U2OS cells (American Type Culture Collection) that have been transduced using CellLight Nucleus-GFP *BacMam 2.0*. (Note: Cells can also be spun down and resuspended in medium or PBS).</li>
 <li>To stain dead cells with PI, transfer 100 &mu;L of the cells to a new microcentrifuge tube. Note that a concentration of 1 x 105 to 1 x 107 cells/mL is recommended for this assay, though the concentration does not have to be exact.</li>
 <li>Next, to stain the dead cells, add 1 &mu;L of the PI-based Tali Dead Cell Red reagent. Vortex briefly to mix. Incubate the Tali Dead Cell Red reagent and cell mixture at room temperature in the dark for 1-5 minutes.</li>
 <li>Following the incubation, immediately proceed to analysis of the sample on the Tali Image-Based Cytometer.</li>
</ol>
2. Performing a Cell Viability Assay Using a Tali Image-Based Cytometer
<ol>
 <li>The Tali cytometer uses the disposable plastic Tali Cellular Analysis Slides that specifically fit into the cytometer and can hold two 25&mu;L samples in separate enclosed chambers (A and B). When handling the slide, be sure to always hold it by the sides.</li>
 <li>To load the slide, pipette 25 &mu;L of sample slowly into the half moon-shaped sample loading area, taking care to avoid forming bubbles or back splatter. Do not over or under fill.</li>
 <li>Here, control cells (unstained, non-transduced) are loaded in chamber A and the stained GFP-expressing cells are loaded in chamber B. The control sample will help determine the level of autofluorescence when analyzing the data.</li>
 <li>To perform a viability assay, touch GFP/RFP on the home screen of the cytometer. The assay options will then appear on the right. Select GFP + Viability.</li>
 <li>Next, to name the sample series, press Name Now. Then, using the touch screen, type the name of the sample series, and press Save. (Note: Select Name Later to automatically assign a name for each sample series by date and time.)</li>
 <li>After naming the sample, the Tali cytometer will automatically advance to the Measure screen. Press the Background tab on the bottom right half of the Measure screen.</li>
 <li>Next, with the control sample (A) facing away from the cytometer, insert the slide in the loading port until it stops. Do not apply force.</li>
 <li>On the background screen, touch Press to Insert New Unstained Cell Control. The slide will automatically be pulled into the cytometer and a live image of the cells will be displayed on the screen.</li>
 <li>Using the focus knob, bring the cells into the proper plane of view. The cells should be uniformly dark with a bright halo around each cell (Figure 1, Left).</li>
 <li>Cells may be undercounted when the transition between the background and the edges of the cells is fuzzy, so that cell boundaries are not well defined (Figure 1, Center). Cells may be overcounted when they have bright centers and dark perimeters (Figure 1, Right).</li>
 <li>To better view the cell population while focusing, slide the red Zoom indicator button to 4X or 16X.</li>
 <li>Once the cells have been brought into focus, touch Press to Run Unstained Cell Control to measure the background fluorescence.</li>
 <li>The cytometer will automatically capture and analyze the images of the sample. It takes about 1 minute to capture and analyze the images in the default (9 image) mode.</li>
 <li>Thumbnails of each field of view are displayed as they are captured and the progress bar provides an ongoing update. After image capture is complete, the cytometer automatically ejects the slide and provides the data from the analysis of the captured images.</li>
 <li>The stored data will be displayed in the drop down menu of the Background tab on the cytometer. The background control sample will be assigned the same name as that given to the experiment and will be imported in the drop down menu. Note: If a background control is not run, the cytometer automatically assigns a fluorescence threshold. This can be manually changed after performing the assay.</li>
 <li>To run the PI-stained transduced sample, remove the slide from the cytometer, flip it around and reinsert it with the transduced sample facing away from the instrument.</li>
 <li>Press the Sample tab, and then touch Press to Insert New Sample. When the image appears on the screen, use the image adjustment knob to bring the cells into focus as before. Then touch Press to Run Sample.</li>
 <li>After image capture is complete, the data from the analysis appears under the Sample tab and the cytometer automatically ejects the slide.</li>
 <li>Appropriately dispose of the Tali Cellular Analysis Slide as biohazardous waste. Tali Cellular Analysis Slides cannot be reused.</li>
</ol>
3. Setting Thresholds and Gates
<ol>
 <li>Once the sample has run, thresholds can now be applied to the sample to home in on a specific cell population. Here, we will adjust the thresholds to determine the percentage of live and dead cells in GFP-transduced cells.</li>
 <li>Under the Sample tab, the number and percentage of cells expressing GFP, live and dead cells expressing GFP, total viability, average cell size, number of cells counted, and cell concentration are displayed.</li>
 <li>In addition, histogram plots displaying cell size, green fluorescence, and red fluorescence (PI) are displayed at the bottom of the screen.</li>
 <li>To set the cell size gate, touch the Cell Size thumbnail to open the cell size histogram. While cultured cells rarely have debris, other samples may contain debris that is larger or smaller than the cells.</li>
 <li>To exclude this debris, slide the blue buttons on the slider bar to exclude both large and small events. Then, touch Apply to re-analyze the data and update the results.</li>
 <li>Next, to add the GFP fluorescence to the analysis, touch the GFP histogram thumbnail to open it. Slide the blue bar to set the threshold just to the right of the dimmest peak, using the control sample as a guide.</li>
 <li>This allows the analysis to include the GFP positive cells, but exclude autofluorescent cells. Autofluorescence is frequently observed when biological molecules within cells are excited.</li>
 <li>Touch Apply to confirm and return to the previous screen. The data displayed under the Sample tab should now reflect the gates and thresholds set for both fluorescence channels.</li>
 <li>Next, to separate the PI stained cells from autofluorescence, press the PI histogram thumbnail to open the PI histogram.</li>
 <li>Again, the control sample peak will be displayed as a gray peak on the histogram. The red peak is the sample fluorescence. The background fluorescence is displayed as a peak closest to the 0 RFU value on this cytometer.</li>
 <li>To eliminate the background fluorescence in the PI channel, move the blue button on the slider bar to adjust the threshold to exclude the peak representing background fluorescence, using the gray peak from the control sample as a reference. Touch Apply to confirm and return to the previous screen.</li>
 <li>Next, to visually confirm that each cell is properly assigned, select a captured field of view for review by pressing the thumbnail of the image in the Zoom tab, then pressing the Layers tab.</li>
 <li>Next, press the GFP or PI button, to display the image captured through that particular channel. Press the same button again to remove the layer from the display; or, to superimpose layers, touch the Multiple Layer button.</li>
 <li>To identify how the cells are counted through a particular channel, touch Circles. The cells that were counted through only the bright-field channel, which do not express fluorescence will be circled in blue. This indicates that that particular cell is live (i.e. PI negative).</li>
 <li>Cells expressing GFP will be seen in the GFP channel, and will be circled in green. Dead cells stained with Dead Cell Red will be seen in the PI channel, and will be circled in red.</li>
 <li>Cells counted in both the red and green channel will be circled in yellow, this indicates that the cell expresses GFP, but is also stained with PI and therefore is dead.</li>
 <li>Occasionally, black circles will appear on the screen, these are objects that have been identified, but have been excluded from the analysis based on cell size.</li>
 <li>Continue to fine tune the threshold on the fluorescence histogram using the steps just described until each cell that is expressing fluorescence is circled with the appropriate color.</li>
 <li>All analysis parameters are automatically saved under the data tab. The Tali cytometer can store data files from approximately 500 sample runs.</li>
 <li>Each file stored in the data tab is available for reanalysis. By selecting the file from the Data tab it will be opened and all histograms and layers become active.</li>
 <li>To archive data and generate reports, the data stored in the cytometer may be transferred to a computer using a USB drive.</li>
</ol>
4. Representative Results:
The Tali Image-Based Cytometer was used to measure the fluorescent protein expression level in cells that were transduced with a nuclear-targeted GFP BacMam 2.0 expression construct. Four representative cell types were transduced (U2OS, 293MSR, HEKn, and CHO-S). For these cell lines, the number of cells that were expressing GFP was reported by the Tali cytometer and compared with data from the same samples analyzed on a flow cytometer. Additionally, cells were stained with Dead Cell Red reagent using the Tali Viability Kit to identify the dead cell population both on the Tali cytometer and on a flow cytometer.
The graphical representations in Figure 2 show the percent of the total population for each cell type that were expressing GFP. These data demonstrate that the Tali cytometer produces data comparable to data generated by a flow cytometer.
<img alt="Figure 1" src="/files/ftp_upload/3659/3659fig1.jpg" /> 
Figure 1. Cells should be properly focused prior to analysis. Tali Cellular Analysis slides with cell concentrations appropriate for analysis (1 x 105 to 1 x 107 cells are recommended). (LEFT) In-focus cells are uniformly dark throughout the cell, with a bright halo around each cell. (CENTER) Cells may be undercounted when the transition between the background and the edges of the cells are fuzzy and the cells have undefined boundaries (RIGHT) Cells maybe overcounted when they have bright centers and dark perimeters.
<img alt="Figure 2" src="/files/ftp_upload/3659/3659fig2.jpg" /> 
Figure 2. Fluorescent protein expression data from the Tali Image-Based Cytometer is comparable to that obtained using flow cytometry. A side-by-side comparison of calculated GFP-expression results in 4 different cell lines in viable cells.

<ol>
	<li>Godfrey, W. L., Hill, D. M., Kilgore, J. A., Buller, G. M., Bradford, J. A., Gray, D. R., Ignatius, M. J., Janes, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementarity+of+Flow+Cytometry+and+Fluorescence+Microscopy.">Complementarity of Flow Cytometry and Fluorescence Microscopy.</a> Microsc. Microanal. 11, 246-247 (2005).</li><li>Mittag, A., Pinto, F. E., Endringer, D. C., Tarnok, A., Lenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+analysis+by+open-source+software+for+affordable+cytometry.">Cellular analysis by open-source software for affordable cytometry.</a> Scanning. 33, 33-40 (2011).</li><li>Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+green+fluorescent+protein+and+its+mutants+in+quantitative+fluorescence+microscopy.">Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.</a> Biophys. J. 73, 2782-2790 (1997).</li><li>Smyth, P. G., Berman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+apoptosis:+methods+for+elucidating+the+mechanism+of+apoptotic+cell+death+from+the+nervous+system.">Markers of apoptosis: methods for elucidating the mechanism of apoptotic cell death from the nervous system.</a> Biotechniques. 32, 648-654 (2002).</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labelled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.</a> J. Immunol. Methods. 184, 39-51 (1995).</li><li>Koopman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+for+flow+cytometric+detection+of+phosphatidylserine+expression+on+B+cells+undergoing+apoptosis.">Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.</a> Blood. 84, 1415-1420 (1994).</li><li>Tanke, H. J., van der Linden, P. W., Langerak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+fluorochromes+to+ethidium+bromide+for+automated+read+out+of+cytotoxicity+tests.">Alternative fluorochromes to ethidium bromide for automated read out of cytotoxicity tests.</a> J. Immunol. Methods. 52, 91-916 (1982).</li><li>King, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+dead+cells+and+measurement+of+cell+killing+by+flow+cytometry.">Detection of dead cells and measurement of cell killing by flow cytometry.</a> J. Immunol. Methods. 243, 155-166 (2000).</li><li>Jones, K. H., Kniss, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propidium+iodide+as+a+nuclear+counterstain+for+immunofluorescence+studies+on+cells+in+culture.">Propidium iodide as a nuclear counterstain for immunofluorescence studies on cells in culture.</a> J. Histochem. Cytochem. 35, 123-125 (1987).</li><li>Dell'Arciprete, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+expression+gene+cloning+by+flow+cytometry.">High-efficiency expression gene cloning by flow cytometry.</a> J. Histochem. Cytochem. 44, 629-640 (1996).</li><li>Lamm, G. M., Steinlein, P., Cotten, M., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+quantitative+and+inexpensive+method+for+detecting+apoptosis+by+flow+cytometry+in+transiently+transfected+cells.">A rapid, quantitative and inexpensive method for detecting apoptosis by flow cytometry in transiently transfected cells.</a> Nucleic. Acids. Res. 25, 4855-4857 (1997).</li><li>Perfetto, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amine+reactive+dyes:+an+effective+tool+to+discriminate+live+and+dead+cells+in+polychromatic+flow+cytometry.">Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.</a> J. Immunol. Methods. 313, 199-208 (2006).</li></ol>

Krissy Remple and Laurel Stone are employees of Life Technologies who produced the instrument used in this article. Life Technologies has sponsored the production of this video-article.

We have just detailed a procedure for performing viability counts and assessing GFP expression using the Tali Image-Based Cytometer. Using the same procedure, this cytometer is capable of performing a number of other assays including: apoptosis, RFP and cell viability analysis using non-transduced cells.
Fluorometric assays of cell viability, gene expression and apoptosis have become key methodologies in biomedical research7-12. In the past, separate instrumentation has been used to obtain quantitative and visual information about cells. While quantitative information about cells within a heterogeneous population has been garnered through the use of flow cytometry, visual or qualitative information has been gathered through the use of fluorescence microscopy 1-4. Here, we present a technology that bridges the gap between population and individual cell studies. Using the Tali Image-Based Cytometer, quantitative data and visual cell data about individual cells or cell populations can be collected simultaneously. The Tali instrument can be used for a range of applications including assessment of gene expression, apoptosis assays, studies of drug efficacy, and assessment of disease progression.
The Tali Image-Based Cytometer captures and analyzes bright-field, red fluorescence and green fluorescence images, allowing information about subpopulations of cells to be obtained. For example, in this assay, we were able to distinguish dead cells from live cells within the GFP-expressing cell population using Dead Cell Red dye. This gives accuracy in the measurement of viable cells expressing GFP, and identifies the population of cells useable for downstream processing. It is important to note that the dead cells in the population could arise from various sources, including normal cell death in culture or cell death caused by environmental conditions (e.g., trypsinization or death induced by the transfection/transduction method chosen).
Here we demonstrate the use of the Tali cytometer with U2OS cells. Similar methods can be performed using most mammalian and insect cells. For each cell line, the Tali cytometer produced quantitative GFP expression data, which is not possible with a visual inspection of the cells using a standard fluorescence microscope. Although these results are comparable to flow cytometry, they are collected in a fraction of the time. The cytometer is capable of accurately counting cells between 5 &mu;m to 60 &mu;m in diameter, ideally at a concentration of 1 x 105 to 1 x 107 cells/mL.
It's important to point out that most standard staining protocols, including the Dead Cell Red staining protocol described here, use propidium iodide to distinguish live and dead cells. This presents a challenge when assessing viability in cells that have been permeabilized for staining of intracellular markers, since PI will stain any cell with a compromised membrane. Since any fluorescent dye or protein that can be detected with the green or red fluorescence channels can be analyzed on the Tali Image-Based Cytometer, future studies may explore the use of alternate dyes, such as amine reactive viability dyes4. A 2006 study by Perfetto et al.12 found these dyes to be easy to use and reproducible in discriminating live and dead cell populations.


Note that the Tali has limitations with respect to the types of assays it can perform. For example, since the objective used by the instrument is 4X, counting operations are limited to larger cell types: sub-cellular structures such as mitochondria or vesicles, and bacterial cells cannot be quantified. Also note that the detection channels in Tali are limited to fluorophores that will excite and emit within the channels. While YFP and bright mCherry signals can be detected, dimmer mCherry signals cannot. Finally, the Tali only analyzes cells in suspension. It will not accurately count cells adhering to a slide. Moreover, while there is only limited capability for counting cells that are irregularly shaped, including some yeast and primary cells. 

In addition, future studies will likely address the use of this system in assessing the expression of intracellular markers. Individual dyes can be checked for spectral overlap with adjacent channels, using Invitrogen's online SpectraViewer tool (<a href="http://www.invitrogen.com/spectraviewer">www.invitrogen.com/spectraviewer</a>). Given the newness of this technology, the full range of applications has yet to be discovered.
In summary, the Tali Image-Based Cytometer demonstrated here presents a new technology that allows for simultaneous visualization and analysis of cell populations, enabling assessment of individual cells as well as cell populations in a compact format with a simple workflow.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Tali Image-Based Cytometer</td>
			<td>Invitrogen</td>
			<td>T10796</td>
		</tr>
		<tr>
			<td>Tali Cellular Analysis Slides, 50 Slides</td>
			<td>Invitrogen</td>
			<td>T10794</td>
		</tr>
		<tr>
			<td>Tali Cellular Analysis Slides, 500 slides</td>
			<td>Invitrogen</td>
			<td>T10795</td>
		</tr>
		<tr>
			<td>Tali Viability Kit - Dead Cell Red</td>
			<td>Invitrogen</td>
			<td>A10786</td>
		</tr>
		<tr>
			<td>Tali Viability Kit - Dead Cell Green</td>
			<td>Invitrogen</td>
			<td>A10787</td>
		</tr>
		<tr>
			<td>Tali Apoptosis Kit </td>
			<td>Invitrogen</td>
			<td>A10788</td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

assessment of gfp expression and viability using the tali image-based cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

Watch this Scientific Journal Video about Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer at JoVE.com

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

assessment-gfp-expression-viability-using-tali-image-based

Research

JoVE Journal

Biology

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.