This research was supported by National Institutes of Health grant (R01-DE025985) and by an American Cancer Society Research Scholar Award (RSG-13-383-MPC). This work was also supported by grants from the U.S. Department of Agriculture (2015-67015-23268 and 2014-67015-21787) and New Research Initiative Grant from University of California, Davis.

CAUTION: Cell lines used in this procedure contain infectious viruses, be careful and only proceed in level 2 BSL facility or higher.
1. Cell Preparation and Maintenance
<ol>
	<li>Culture TREx-K-RTA BCBL-1 cells (or another KSHV infected PEL cell lines) in RPMI1640 medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin streptomycin glutamine solution.</li>
	<li>Grow cells at 37 &#176;C with 5% carbon dioxide (CO2), making sure to continuously grow and to split cells by a ratio of 1:4 every 2&#8211;4 days.</li>
	<li>Monitor cell cultures with a light microscope every day to confirm good cell growth and morphology, otherwise it is recommended to restart the cell culture or to adjust the cell culture conditions accordingly.</li>
</ol>
2. Thymidine Synchronization and Lytic Induction
<ol>
	<li>In a 10 cm Petri dish, plate TREx-K-Rta BCBL-1 cells in freshly prepared media (1 x 106 cells/2 mL of media)</li>
	<li>Add 200 mM thymidine (final concentration: 2 mM) to the dish, mix and incubate for 18 h.</li>
	<li>Centrifuge the cells for 5 min at 500 x g. Wash the cells with sterile 1x phosphate-buffered saline (PBS), centrifuge again, and resuspend cells in freshly prepared media. Repeat for a total of 3 washes. Resuspend in freshly prepared media.</li>
	<li>Allow the cells to exit S-phase for 8 h.</li>
	<li>Add thymidine (final concentration: 2 mM), mix and incubate for 16 h.</li>
	<li>Centrifuge the cells for 5 min at 500 x g. Wash the cells with sterile 1x phosphate-buffered saline (PBS), centrifuge again, and resuspend cells in freshly prepared media. Repeat for a total of 3 washes. Resuspend in freshly prepared media.</li>
	<li>To induce viral reactivation, add 12-O-tetradecanoyl-phrobol-13-acetate (TPA, final concentration 20 ng/mL) and doxycycline (DOX, final concentration 100 ng/mL) to the cell culture, mix and incubate for 4 h. For cell lines without a K-Rta inducible cassette, stimulate reactivation using TPA (final concentration 20 ng/mL and sodium butyrate (1 mM)).</li>
	<li>Centrifuge the cells for 5 min at 500 x g. Wash the cells with sterile 1x phosphate-buffered saline (PBS), centrifuge again, and resuspend cells in complete culture media. Repeat for a total of 3 washes. Resuspend in complete culture media.</li>
	<li>Allow the cells to grow for 24 h.</li>
</ol>
3. Fixation and Permeabilization
NOTE: Before proceeding, ensure fixing solution (3.7% formaldehyde in DEPC-treated PBS), diethyl pyrocarbonate-treated phosphate-buffered saline (DEPC-PBS), glycine DEPC PBS (final concentration of glycine: 100 mM) and permeabilizating solution (50% acetone, 50% methanol) are prepared. Typically, each slide will require at least 0.5 million cells, multiply if necessary and include extra cells for back up especially if cells are not healthy.
<ol>
	<li>Collect cells into a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge the cells for 2 min at 200 x g. Wash the cells 3 times with 1 mL of sterile DEPC PBS. Resuspend in 1.2 mL of DEPC PBS.</li>
	<li>Place the appropriate number of coverslips (determined by the experimental setup) into the bottoms of a 6-well plate.</li>
	<li>Pipette 200 &#181;L of the cell DEPC PBS mixture onto each coverslip, ensure that at least 0.5 million cells are on each slide. Allow the cells to settle for 2 min, and aspirate excess DEPC PBS leaving only a thin layer of cells. It is normal for cells to be removed in this process. 
	Caution: PFA is toxic, wear appropriate protection.</li>
	<li>Carefully add 1 mL of fixing solution (3.7% formaldehyde in DEPC PBS) to each cover slip. Allow the cells to fix for 10 min.</li>
	<li>Wash coverslips 3 times with DEPC PBS.</li>
	<li>Carefully add 1 mL of glycine DEPC PBS (final concentration of glycine: 100 mM) to each cover slip. Allow the cells to quench for 5 min.</li>
	<li>Add 1.5 mL of DEPC PBS and either place on a shaker for 5 min or shake firmly but gently in hand for 1 min. Aspirate the excess DEPC PBS. Be careful not to disrupt the cells. Repeat this step for a total of 3 washes.</li>
	<li>Carefully add 1 mL of permeabilizing solution (50% acetone, 50% methanol) to each cover slip. Allow the cells to permeabilize for 15 min. 
	NOTE: At this point, the coverslips can be stored in a -20 &#176;C freezer for no longer than a week. Avoid freezing or minimize the time spent in the freezer as image quality can degrade quickly. Make sure that when in the freezer that the cells remain wet and covered in methanol/acetone, and to ensure that they properly rehydrate with DEPC PBS after removal from the freezer.</li>
	<li>Add 1.5 mL of DEPC PBS and either place on a shaker for 5 min or shake firmly but gently in hand for 1 min. Aspirate the excess DEPC PBS, be careful not to disrupt the cells. Repeat this step for a total of 3 washes.</li>
</ol>
4. IFA and RNA-FISH
NOTE: Regarding RNA FISH Probe labeling and preparation: For the K-Rta intron which is 959 base pairs, 31 different probes were generated that were 20 base pairs each, containing a GC content of about 50%. Working and stock solutions of the probes were aliquoted into 100 &#181;M and 2.5 &#181;M concentrations, respectively.
<ol>
	<li>Label microscope slides with necessary experimental information or some sort of distinguishable label.</li>
	<li>Line the bottom of a sealable plastic container with a moist paper towel.</li>
	<li>Prepare the primary antibody solution (1.5 &#181;L of LANA antibody, 0.75 &#181;L of RNA Pol II antibody, 3 &#181;L of baker&#39;s yeast tRNA, DEPC PBS up to 30 &#181;L), and multiply recipe for each coverslip present. Place 30 &#181;L of primary antibody solution onto each labeled microscope slide.
	<ol>
		<li>Titrate the primary antibody of interest first and use the optimal amount. Be sure to use purified IgG, as serum (ascites fluid) may contain large amount of RNase.</li>
	</ol>
	</li>
	<li>Place the coverslip (cells facing the solution) onto the labeled microscope slides and move the slides into the container with the moist paper towel. Be careful not to introduce bubbles. Cover the exterior of the container with plastic wrap. Move the container into an incubator set to 37 &#176;C for 1 h.</li>
	<li>Prepare a FISH wash buffer (2x saline sodium citrate buffer (SSC) (final), 10% formamide, 90% DEPC dH2O).</li>
	<li>Prepare 2x Hybridization Buffer (4x SSC, 20% dextran sulfate).</li>
	<li>Prepare the secondary antibody and FISH intron probe buffer (20 &#181;L of 2x hybridization buffer (final 1x Hyb Buffer), 4 &#181;L of baker&#39;s yeast tRNA (final 10%), 4 &#181;L of formamide (final 10%), intron K-Rta probe (final 125 nM), 0.8 &#181;g of secondary antibody, DEPC dH2O up to 40 &#181;L). Multiply recipe for every slide needed.</li>
	<li>After incubation, wash cells 3 times with DEPC PBS in a 6 well plate for 5 min each.</li>
	<li>Wash with FISH wash buffer 3 times for 5 min each. Keep coverslip in FISH-wash buffer until finish preparing mixture of secondly antibody and intron probe (step 4.7).</li>
	<li>Clean glass slides used for primary antibody incubation for hybridization.</li>
	<li>Place 40 &#181;L of the secondary antibody and FISH intron probe containing solution onto each glass slide.</li>
	<li>Place the coverslips with the cells onto the slides with the cell side facing down on top of the buffer, be careful not to introduce bubbles.</li>
	<li>Place the slides into a plastic container with a moist paper towel on the bottom. Wrap the plastic container in first plastic wrap and then aluminum foil.</li>
	<li>Incubate the container with the slides at 37 &#176;C for 16-24 h.</li>
	<li>After incubation, carefully slide the coverslip from the edge of the glass slide, and put back into 6 well plates facing up, and wash cells with FISH-wash buffer 3 times in a 6 well plate for 5 min each.</li>
	<li>Wash the cells 2 times with 2x SSC.</li>
	<li>Add DAPI (1:1,000) into the 2x SSC and allow to sit for 5 min at room temperature.</li>
	<li>Wash the cells 2 times with 2x SSC.</li>
	<li>Clean the glass slides used for hybridization, and add 10 &#181;L of mounting solution onto the glass slides.</li>
	<li>Place the coverslips face down onto the mounting solution. Be careful not to introduce bubbles.</li>
	<li>With a laboratory tissue, gently remove the excess mounting solution, being careful not to squash the cells.</li>
	<li>Using nail polish, apply an ample layer around the edge of the coverslips and allow to dry.</li>
	<li>Proceed to perform fluorescence microscopy.</li>
</ol>
5. 3D Fluorescence Microscopy
NOTE: While protocols will vary with the type of fluorescence microscope system used, the following steps will help to ensure the acquisition of optimal image data for quantitative analysis.
<ol>
	<li>Pretreat fixed samples and mount in anti-fade media to delay fluorescence photobleaching during imaging.</li>
	<li>Use a high quality objective (such as a 60X 1.42 N.A oil-immersion lens), which can capture a whole cell (or cell nucleus) in the field-of-view.</li>
	<li>Set exposure parameters (e.g., excitation power, exposure time) for each fluorescence color channel, using positive- and negative-control samples. Acquire fluorescence images. 
	NOTE: Positive control samples that contain only one fluorescent label should also be used to determine the level of &#34;crosstalk&#34; between color channels. For imaging fixed cell specimens, fluorescence excitation power can be reduced (with slightly longer exposure times) to minimize photobleaching.</li>
	<li>Once the imaging protocol has been established, maintain the same exposure parameters consistent for all samples in a study &#8212; so that image data can be accurately analyzed and compared.</li>
	<li>Perform post-acquisition image processing and 3D reconstruction. Identify the K-Rta transcripts by RNA-FISH signal (red), LANA molecules by immunofluorescence (green), and nuclear chromatin (where applicable) stained with DAPI (blue). 
	NOTE: Regions of the cell nucleus, in which K-Rta and LANA are co-clustered, are thus believed to be associated with viral transcription factories and replication complex.</li>
</ol>

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The authors of this paper have no financial ties or competing conflicts of interests.

There are some aspects of the protocol that can be altered to accommodate unusual circumstances. The choice of fixation and permeabilization buffers can also be altered. For fixation buffers, paraformaldehyde is also effective and ethanol can be used for permeabilization. 
Quenching the formaldehyde with glycine PBS is recommended as it prevents formaldehyde from disabling the antibodies in absence of bovine serum albumin (BSA), but if the coverslips are washed thoroughly enough, the glycine P...

It has become increasingly clear that the spatiotemporal organization of the nucleus plays an important role in modulating finely-tuned gene expression in eukaryotes. Most tissue specific genes are distributed over many chromosomes and need to be regulated synchronously in order to respond to specific stimuli in a coordinated manner1. Cells construct active chromatin hubs (ACH) as a way of bringing together genes and their cis-regulatory components to a specific nuclear space1.
It has also been demonstrated by various studies that although the nucleus seems very dense and viscous, biologically active molecules can traverse the nucleus rather quickly via diffusion2. As a consequence of this ephemeral property, most DNA binding proteins &#39;jump&#39; from binding site to binding site, feeling their way around the nuclear space, which allows for a highly adaptive and versatile nucleus2.
Despite this dynamic behavior of biomolecules within the nucleus, nuclear bodies without membranes such as (but not limited to) the nucleolus, Cajal bodies, and promyelocytic leukemia nuclear bodies (PML-NB) still exist. It is through a variety of mechanisms such as tandem DNA repeats (nucleolus), rRNA (nucleolus) and structural proteins such as coilin (Cajal bodies) or PML proteins (PML-NB) that hold these constructs together3,4,5. These structures and other congregations such as transcription factories serve as scaffolds that not only increase the local concentration of required components, but also regulate the composition of proteins and nucleic acids within them to ultimately create a central site for efficient cellular functions6.
Visualizing when and where nuclear structures form provides a wealth of information to researchers studying epigenetics. From a virology perspective, the reactivation of latently infected viruses, such as KSHV, significantly alters the landscape of the nucleus and distribution of nuclear enzymes to shift transcription primarily from cellular genes to viral genes, ultimately to produce fully functional viral progeny7,8. How does KSHV manipulate the cellular gene expression machinery to facilitate viral gene expression? Such information could also shed light on temporal cellular gene regulatory mechanisms.
Like all other herpesviruses, KSHV has two life cycles called lytic replication and latency. KSHV primarily resides in the latency stage, in which most of its viral gene expression is silenced, except latency associated genes9,10. During latency KSHV produces latency associated nuclear antigen (LANA), which constitutively binds the viral genomes and tethers viral chromatin to the human chromosome11. Because of LANA&#39;s intimate relationship with the viral genome, it is possible to use IFA and DAPI to stain and locate where the viral episomes were in relation to the host chromatin.
To study KSHV reactivation at the single episome level and the association with other viral episomes in an infected cell, a strategy to locate actively transcribing viral chromatin in situ has been established. Accordingly, LANA and RNAPII IFA with intron RNA-FISH were combined, by generating RNA-FISH probes that bind to the intron region (exact probe sequences can be found in the Izumiya lab&#39;s most recent publication8) of KSHV K-Rta-the key viral protein that is essential and sufficient for KSHV reactivation-it was possible to identify where transcription was actually taking place8,11,12,13,14,15. This intron RNA-FISH technique enables researchers to visualize where mRNA is being transcribed immediately before it is spliced and exported to cytoplasm16,17.
KSHV can be reactivated by various chemical stimuli including phorbol esters such as 12-O-Tetradeconoyl-phorbol-13acetate (TPA) and histone deacetylase inhibitors such as sodium butyrate, additionally KSHV can be induced to reactivate by overexpression of the viral transcription factor, K-Rta19. Researchers have successfully increased the efficiency of KSHV reactivation by synchronizing the cell&#39;s cycles prior to inducing reactivation18. Thus, for these particular studies, cells were synchronized using a double thymidine block (protocol described below) and incubated with TPA and doxycycline (Dox) for a short time. Doxycyline was used because the cell line utilized in these experiments has a doxycycline-inducible K-Rta cassette, which was cloned from cDNA and does not include the intron region of K-Rta. Although it is possible to reactivate KSHV using only induced K-Rta expression, it has been proven by other researchers that due to a variety of different biochemical factors K-Rta expression alone proves to be a feeble reactivation stimuli20. By combining all of these, and by limiting the drug incubations to a short period of time, a robust but not overly artificial KSHV reactivation was achieved for imaging.
After labeling LANA, RNAPII, K-Rta introns, and DNA as described in this paper, 3D fluorescence imaging was performed using a widefield deconvolution microscope. After processing with imaging software, the spatial distribution of active viral episomes can be properly evaluated. Using this technique, the central questions regarding the fundamental nature of the formation of active chromatin hubs and other nuclear structures can be studied. Having identical viral episomes in a single cell that processes the same regulatory elements may represent a unique research tool to deepen the understanding of spatiotemporal gene regulatory mechanisms.
A limitation of imaging multiple cell specimens fixed at different time points to characterize an inherently dynamic molecular process is that subtle or small-scale changes in fluorescence distribution are undetected or deemed insignificant. This is true unless in the rare instance, every cell observed displays the same subtle change. Thus, the full spatiotemporal relationship of active viral transcription and other nuclear structures cannot be critically evaluated using fixed imaging. To address these technical challenges, the best approach is to image live cells that have marked viral episomes and to follow the location of key cellular enzymes over time.

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">RPMI medium 1650 GlutaMAX</td>
			<td style="width:71px;">Gibco by Life Technologies</td>
			<td style="width:119px;">61870-036</td>
			<td style="width:167px;">Needs to be warmed to 37 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Omega Scientific Inc.</td>
			<td style="width:119px;">FB-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Pen Strep Glutamine (100x)</td>
			<td style="width:71px;">Gibco by Life Technologies</td>
			<td style="width:119px;">10378-016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Thymidine&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">T9250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dulbecco&#8217;s Phosphate Buffered Solution</td>
			<td style="width:71px;">Gibco by Life Technologies</td>
			<td style="width:119px;">14190-144</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">TPA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">P1585</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium Butyrate&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">B5887</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Formaldehyde Solution&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">252549</td>
			<td style="width:167px;">Corrosive, toxic, health hazard</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">D-5758</td>
			<td style="width:167px;">Acute toxicity&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glycine</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">410225</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acetone</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">179124</td>
			<td style="width:167px;">Flammable, toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Methanol</td>
			<td style="width:71px;">Fisher Chemical&#160;</td>
			<td style="width:119px;">67-56-1</td>
			<td style="width:167px;">Flammable, toxic, health hazard</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Rat monoclonal antibody to KSHV/HHV-8 ORF73</td>
			<td style="width:71px;">Advanced Biotechnologies</td>
			<td style="width:119px;">13-210-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-RNA Polymerase II Antibody clone CTD4H8</td>
			<td style="width:71px;">EMD Milipore</td>
			<td style="width:119px;">05-623</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ribonucleic acid, transfer from baker&#8217;s yeast (S. cerevisiae)&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">9014-25-9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Saline sodium citrate buffer 20x (SSC)</td>
			<td style="width:71px;">Thermo Fisher Scientific&#160;</td>
			<td align="right" style="width:119px;">15557044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Formamide&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">295876</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Omnipur Dextran Sulfate, 50% solution</td>
			<td style="width:71px;">EMD Milipore</td>
			<td style="width:119px;">1916C093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">DAPI (4&#8217;,6-diamindino-2-phenylindole, Dihydrochloride)</td>
			<td style="width:71px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">D1306</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">slowFade Gold antifade reagent</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">S36936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Micro cover glass 22x22</td>
			<td style="width:71px;">Matsunami</td>
			<td style="width:119px;">C022221</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">VoloCITY</td>
			<td style="width:71px;"></td>
			<td style="width:119px;">3D imaging software</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DeltaVision microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Superfrost/Plus microscope slides</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-15</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

functional imaging of viral transcription factories using 3d fluorescence microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

A slightly abridged protocol was performed where BCBL-1 cells were used and only LANA and K-Rta RNA were stained (Figure 1). This experiment allows us to examine where actively transcribing viral episomes are and the heterogeneity of response to KSHV reactivation stimuli in a population of cells. In the cell indicated by the arrow in Figure 1, the distinct K-Rta fluorescence in regions that closely match the distribution of viral...

Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

functional-imaging-viral-transcription-factories-using-3d

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JoVE Journal

Biology

7.0K Views.  University of California, Davis. Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

Video: Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...