This work was supported by National Institutes of Health grants GM 100201 to I.F.S, and GM 048071 and GM 065830 to I.P.

We present detailed procedures for imaging local Ca2+ events in human neuroblastoma SH-SY5Y cells. These procedures can be adapted to image intracellular Ca2+ signals in many cell types8-10.
1. Preparation of Cells
<ol>
	<li>Culture the cells to be imaged according to instructions listed on their supplier&#8217;s website.</li>
	<li>A few days prior to imaging, harvest cells grown in a tissue culture flask using 1 ml of 0.25% Trypsin/EDTA (2-3 min). Following detachment, add an equal volume of culture media to inactivate the trypsin.</li>
	<li>Perform a cell count using a hemocytometer and seed cells into glass-bottom imaging dishes at a density of 3-7 x 104 cells per dish. Select cells for imaging experiments when they reach 60% confluence (2-3 days).</li>
</ol>
2. Preparation of Solutions and Reagents
<ol>
	<li>Prepare a Ca2+-containing HEPES buffered salt solution (Ca2+-HBSS) composed of (mM): 135 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose; pH=7.4 at room temperature (RT) with NaOH. Prepare Ca2+-HBSS without the addition of glucose and store at 4 &#176;C; add glucose to the solution immediately prior to use.</li>
	<li>Solubilize membrane-permeant forms of the fluorescent Ca2+ indicator Cal-520 (1 mM), ci-IP3 (200 &#181;M), and EGTA (100 mM) in dimethyl sulfoxide (DMSO) containing 20% pluronic F-127. Aliquot these reagents into Eppendorf tubes and store, shielded from moisture and light, at -20 &#176;C.</li>
</ol>
3. Loading Cells with Membrane-permeant Cal-520, ci-IP3 and EGTA
<ol>
	<li>Prepare the &#8220;loading buffer&#8221; by diluting stock solutions of membrane-permeant Cal-520 and ci-IP3 to a final concentration of 5 &#181;M and 1 &#181;M, respectively, in Ca2+-HBSS.</li>
	<li>Aspirate culture media and rinse cells by replacing media with Ca2+-HBSS three times.</li>
	<li>Remove Ca2+-HBSS and incubate cells in loading buffer for 60 min at RT in the dark.</li>
	<li>Remove loading buffer and rinse cells by replacing media with Ca2+-HBSS three times.
	<ol>
		<li>If desired, at this step, load cells with EGTA/AM by diluting the stock solution to a final concentration of 5 &#181;M in Ca2+-HBSS and then incubate cells for 30-60 min at RT in the dark. Following this incubation, rinse cells three times with Ca2+-HBSS before proceeding to step 3.5.</li>
	</ol>
	</li>
	<li>Incubate cells in Ca2+-HBSS for 30 min to allow for de-esterification of loaded reagents.</li>
	<li>Immediately prior to imaging, replace Ca2+-HBSS with &#8220;fresh&#8221; Ca2+-HBSS in order to remove any dye that might have leaked into the bath during the final incubation.</li>
</ol>
4. Ca2+ Image Acquisition
<ol>
	<li>Place a small drop of immersion oil onto the 100X APO TIRF objective (NA 1.49).</li>
	<li>Mount the imaging dish on the stage of the inverted microscope and bring the cells into focus using transmitted light. It is important to secure the imaging dish to prevent movement during recording.</li>
	<li>Illuminate cells with a 488 nm laser in order to excite Cal-520 and collect emitted fluorescence (&#955; &#62;510 nm) using a high-speed EMCCD camera. 
	NOTE: The software package that operates the microscope allows the user to translate a focusing lens allowing the laser beam to be introduced either at the extreme edge of the objective back aperture (for TIRF excitation) or more centrally (for &#8220;WF&#8221; excitation).</li>
	<li>Using the EMCCD software, reduce the imaging field from the full 512 x 512 pixels in order to collect data at the necessary temporal resolution to capture local Ca2+ events. 
	NOTE: For example, center quad acquisition of 256 x 256 pixels speeds up the acquisition rate to 66 fps. It is also possible to further increase the frame rate of the EMCCD camera to 500 fps using an Isolated Crop Mode function.</li>
	<li>Configure the software to automatically record a few seconds of baseline activity, deliver a UV flash to the cells and continue recording for another 10-30 sec. 
	NOTE: A UV flash is delivered using a Xenon arc light source introduced through a UV reflecting dichroic mirror in the rear port of the microscope. The duration and intensity of the UV flash is adjusted empirically to evoke a desired frequency of local Ca2+ events.</li>
	<li>Save files as image stacks for off-line analysis.</li>
</ol>
5. Automated Ca2+ Image Analysis
NOTE: It is possible to view, process and analyze captured data using many commercial software packages. However, we have developed an algorithm to rapidly automate identification and analysis of local Ca2+ signals. A detailed description of the spatial and temporal filters used in this algorithm, generation of the &#916;F/F0 and identification/analysis routines can be found in11. This algorithm has been developed to run on the open-source software platform Python, and can be obtained, together with sample experimental data and detailed user instructions, by e-mailing the corresponding author (ismith@uci.edu).
<ol>
	<li>Convert files to be imported into the custom-written algorithm to the multi-plane .tiff file format.</li>
	<li>Locate the folder containing the analysis algorithm and double click the run.exe.</li>
	<li>Select the .tiff file to be analyzed.</li>
	<li>Choose analysis parameters in the open dialog box. See Figure 2A for default parameters for the sample data.</li>
	<li>Determine the black level to be offset from the image stack by either moving the cursor to a part of the field of view that does not contain a cell (see Figure 2B) or by manually entering a black level using the &#8216;Set Black Level&#8217; prompt.</li>
	<li>Observe four windows on the screen following analysis by the software (Figure 2C-F). C is a monochrome image of resting fluorescence from cells being analyzed. White squares superimposed on this image are event locations, determined as the centroid positions of two-dimensional Gaussian functions fitted to events.</li>
	<li>Click on each event location or press the cursor keys to cycle between events. Upon doing so the background-subtracted, Gaussian smoothed fluorescence ratio changes (&#916;F/F0) at each of these sites update in window D. 
	NOTE: Red highlighted events are determined to originate from that particular site and not from &#8216;bleed-through&#8217; from activity localized to adjacent sites. The lower blue trace indicates where the fluorescence signal at the selected site exceeded the detection threshold, whereas the black line identifies events originating from that particular site (corresponding to the red highlighted events).</li>
	<li>Click on a red highlighted event to update window E that displays the temporal evolution of the event, and window F which displays the spatial profile of the event averaged over its time course, together with the spatial profile of the fitted Gaussian function.</li>
	<li>Manually review the events that have been identified so that artifacts can be rejected from analysis. Delete such events by right clicking the red highlighted events. 
	NOTE: In our hands, Ca2+ flux through single IP3R channels evokes signals with &#916;F/F0 about 0.11, so any detected &#8216;events&#8217; appreciably smaller than this are likely to be false positives.</li>
	<li>Export data by selecting &#8216;Save to Excel&#8217; or export data on a per cell basis by drawing around the cell of interest and selecting &#8216;Save Cell&#8217;. 
	NOTE: Data are saved to the same folder as the original image stack.</li>
</ol>

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1 (1), 11-21 (2000).</li><li>Hill-Eubanks, D. C., Werner, M. E., Heppner, T. J., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+smooth+muscle.">Calcium signaling in smooth muscle.</a> Cold Spring Harb Perspect Biol. 3 (9), a004549 (2011).</li><li>Hagenston, A. M., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+synapse-to-nucleus+communication.">Calcium signaling in synapse-to-nucleus communication.</a> Cold Spring Harb Perspect Biol. 3 (11), a004564 (2011).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elementary+and+global+aspects+of+calcium+signalling.">Elementary and global aspects of calcium signalling.</a> J Physiol. 499 (Pt 2), 291-306 (1997).</li><li>Lipp, P., Niggli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hierarchical+concept+of+cellular+and+subcellular+calcium+signalling).">A hierarchical concept of cellular and subcellular calcium signalling).</a> Prog Biophys Mol Biol. 65 (3), 265-296 (1996).</li><li>Parker, I., Yao, Y., Ilyin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics+of+calcium+liberation+induced+in+Xenopus+oocytes+by+photoreleased+inositol+trisphosphate.">Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.</a> Biophys J. 70 (1), 222-237 (1996).</li><li>Minta, A., Kao, J. P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+cytosolic+calcium+based+on+rhodamine+and+fluorescein+chromophores.">Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.</a> J Biol Chem. 264 (14), 8171-8178 (1989).</li><li>Demuro, A., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+activity+and+localization+of+single+voltage-gated+Ca(2+)+channels+by+total+internal+reflection+fluorescence+microscopy.">Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy.</a> Biophys J. 86 (5), 3250-3259 (2004).</li><li>Smith, I. F., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+quantal+substructure+of+single+inositol+trisphosphate+receptor+channel+activity+during+calcium+puffs+in+intact+mammalian+cells.">Imaging the quantal substructure of single inositol trisphosphate receptor channel activity during calcium puffs in intact mammalian cells.</a> Proc Natl Acad Sci U S A. 106 (15), 6404-6409 (2009).</li><li>Reissner, K. J., et al. <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+postsynaptic+mechanism+for+heterosynaptic+sharing+of+short-term+plasticity.">A novel postsynaptic mechanism for heterosynaptic sharing of short-term plasticity.</a> J Neurosci. 30 (26), 8797-8806 (2010).</li><li>Ellefson, K. S., Parker, B., Smith, I., F, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+algorithm+for+automated+detection,+localization+and+measurement+of+local+calcium+signals+from+camera-based+imaging.">An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging.</a> Cell Calcium. , (2014).</li><li>Smith, I. F., Wiltgen, S. M., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+puff+sites+adjacent+to+the+plasma+membrane:+Functional+and+spatial+characterization+of+calcium+signaling+in+SH-SY5Y+cells+utilizing+membrane-permeant+caged+inositol+trisphosphate.">Localization of puff sites adjacent to the plasma membrane: Functional and spatial characterization of calcium signaling in SH-SY5Y cells utilizing membrane-permeant caged inositol trisphosphate.</a> Cell Calcium. 45, 65-76 (2009).</li><li>Shuai, J., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+single-channel+recording+by+imaging+calcium+flux+through+individual+ion+channels:+theoretical+considerations+and+limits+to+resolution.">Optical single-channel recording by imaging calcium flux through individual ion channels: theoretical considerations and limits to resolution.</a> Cell Calcium. 37 (4), 283-299 (2005).</li></ol>

The authors declare that they have no competing financial interests.

We describe here protocols for imaging local Ca2+ events in cultured mammalian cells using fluorescent Ca2+ indicators. Furthermore, we describe an algorithm with an intuitive user interface that automates identification and analysis of acquired data. The procedure described here utilize the fluorescent Ca2+ indicator Cal-520, but many other Ca2+ sensitive dyes such as Fluo-3, Fluo-4, Fluo-8 and Oregon Green BAPTA-1 perform sufficiently well to image Ca2+ microdomain...

Calcium ions (Ca2+) ubiquitously regulate a diverse range of biological processes, including gene expression, secretion and long-lasting changes in synaptic plasticity1. One way through which Ca2+ can act in such a diverse manner is through the different spatial and temporal patterns of Ca2+ signals a cell can generate. For example global elevations in cytosolic [Ca2+] trigger contraction in smooth muscle tissue2 whereas smaller, localized transient elevations (local Ca2+ microdomains) stimulate gene expression essential for learning and memory3.
Free cytosolic [Ca2+] is maintained at ~100 nM at rest, but can rapidly rise to several micro-molar following the influx of Ca2+ into the cytosol through Ca2+-permeable ion channels located in the plasma membrane and by the liberation of Ca2+ from intracellular stores. Our lab focuses on the inositol 1,4,5-trisphosphate receptor (IP3R), which forms a Ca2+ release channel located in the endoplasmic reticulum (ER) membrane. Upon binding of both IP3 and Ca2+ to the cytosolic activating sites of the receptor, the IP3R channel opens to liberate Ca2+ sequestered within the ER lumen. The release of Ca2+ may remain spatially restricted to a small cluster of IP3Rs to generate a local cytosolic microdomain of Ca2+ (Ca2+ puff4) or, depending on the proximity of neighboring clusters of IP3Rs, may propagate throughout a cell by recruiting multiple puff sites through a process of Ca2+-induced Ca2+-release (CICR)5,6.
The introduction of fluorescent small molecule Ca2+ indicator dyes developed by Roger Tsien7, coupled with advanced microscopy imaging techniques, has greatly facilitated our understanding of Ca2+ signaling. Recent advances in cameras used for microscopy now allow for imaging transient local Ca2+ events such as puffs with unprecedented spatial and temporal resolution. Currently available EMCCD cameras enable imaging with 128 x 128 pixels at &#62;500 frames sec-1 (fps) and the new generation of complementary metal-oxide semiconductor (CMOS) cameras provide higher pixel resolution, and even faster speed at the expense of slightly higher noise levels. In conjunction with total internal reflection (TIRF) microscopy it is now possible to image single Ca2+ channel events8,9. This approach allows for the imaging of hundreds of channels/events simultaneously, while generating large data sets (ca. 1Gb per min) that render manual processing, visual identification and analysis impracticable and place an onus on the development of automated algorithms.
Here, we present procedures and protocols for imaging local Ca2+ signals in intact mammalian cells using fluorescent Ca2+ indicators. We further demonstrate an algorithm developed in the open-source environment Python that automates identification and analysis of local Ca2+ events imaged by both TIRF and conventional wide field epi-fluorescence (WF) microscopy. Although we describe these approaches in the context of IP3-generated Ca2+ signals, they are readily amenable to study local changes in cytosolic [Ca2+] emanating from a variety of Ca2+-permeable ion channels located in either the surface membrane or intracellular organelles8-10.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:813px;">Name</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalogue No.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Neuroblastoma SH-SY5Y cells</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cal-520/AM</td>
			<td>AAT Bioquest Inc.</td>
			<td>21130</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ci-IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4.5-dimethoxy)benzyl-myo-Inositol 145-trisphosphate-Hexakis(propionoxymethyl)</td>
			<td>SiChem</td>
			<td>cag-iso-2-145-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO/20% pluronic F127</td>
			<td>Invitrogen</td>
			<td>P-3000MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA/AM</td>
			<td>Invitrogen</td>
			<td>E-1219</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom imaging dishes</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</td>
		</tr>
	</tbody>
</table>

imaging local ca2+ signals in cultured mammalian cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Figure 1A shows a WF image of resting Cal-520 fluorescence in human neuroblastoma SH-SY5Y cells also loaded with ci-IP3. Exposure of these cells to a 100 msec UV flash to photo-release i-IP3 elicited transient Ca2+ puffs at discrete sites (noted by the white circles in Figure 1A). Fluorescence traces measured at these sites showed a rapid rising phase owing to transient openings of IP3Rs, followed by a much slower falling phase (Figure ...

Watch this Scientific Journal Video about Imaging Local Ca2+ Signals in Cultured Mammalian Cells at JoVE.com

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

imaging-local-ca2-signals-in-cultured-mammalian-cells

Research

JoVE Journal

Biology

10.7K Views.  University of California, Irvine. Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Video: Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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 ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.