Name: determination of the relative cell surface and total expression of recombinant ion channels using flow cytometry
Uploaded: 2016-09-28T15:00:00
Description: Watch this Scientific Journal Video about Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry at JoVE.com

We thank Mr. Serge S&#233;n&#233;chal and Dr. Jacques Thibodeau for sharing their expertise and granting us access to their flow cytometry and cell sorting platform. This work was completed with the operating grant 130256 from the Canadian Institutes of Health Research, a grant-in-aid from the Canadian Heart and Stroke Foundation, and support from the "Fondation de l'Institut de Cardiologie de Montr&#233;al" to L.P.

1. Doubly Tagged DNA constructs
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	<li>Insert the HA epitope (YPYDVPDYA) in the extracellular linker of CaV&#945;2&#948;1 between D676 and R677 by site directed mutagenesis (Figure 3B)20. Use Forward primer gccggattatgcgGGAAAACTCCAAACAACC and Reverse primer acatcatacggataTCAATAAATTCATTGAAATTTAAAAGAAATTC.</li>
	<li>Subclone the cDNA sequence of the tagged HA CaV&#945;2&#948;1 into the mammalian pmCherry-N1 expression vector designed to express the protein fused to the...

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This flow cytometry-based assay was successfully applied to the measurement of relative total and cell surface levels of fluorescently-labelled pore-forming and associated subunits of voltage-gated calcium channels14,22,26. It is best used when investigating the impact of genetic mutations and thus requires that the intrinsic fluorescence intensity of the fluorescently-labelled tagged wild-type construct be at least 10 to 100-fold larger than for the fluorescence intensity of the fluorescently-labelled untagge...

This paper provides a reliable assay to report the relative cell surface expression of membrane proteins such as ion channels expressed in recombinant cells using the existing flow cytometry technology. Ion channels are pore-forming membrane proteins that are responsible for controlling electrical signals by gating the flow of ions across the cell membrane. They are classified by the activation mechanism, nature, and selectivity of ion species transiting through the pore where they are localized. At the cellular and tissue levels, the macroscopic ion fluxes through ion channels are the product of biophysical (gating and permeation), biochemical (phosphorylation), and biogenesis (synthesis, glycosylation, trafficking, and degradation) properties1. Each of these processes is unique to every type of ion channels and is optimized to fulfill the physiological role of the ion channel. Consequently, alterations in any of these fine-tuned processes through an inherited or a genetic modification, often referred to as &#34;channelopathy&#34;, can be detrimental to cell homeostasis. It is important to stress that delivering the &#34;right&#34; amount of ion channels at the cell surface is critical to cell homeostasis. Even small increases (gain-of-function) and small decreases (loss-of-function) in ion channel activity have the potential to cause a serious pathology over a lifetime. Defects in the cell surface delivery of mature ion channels is an important determinant in numerous channelopathies, such as cystic fibrosis (CFTR ion channel)2 and cardiac arrhythmias of the long QT syndrome form (cardiac potassium channels)3.
Channelopathies are associated with cardiac sudden death4. The current worldwide prevalence of all cardiac channelopathies is thought to be at least 1:2,000-1:3,000 per individual5 and are responsible for about half of sudden arrhythmic cardiac death cases6. Dysfunction in cardiac voltage-gated sodium-, potassium-, and calcium- selective ion channels are known to play a key role in this process. The L-type CaV1.2 voltage-gated calcium channel is required to initiate synchronized heart muscle contraction. The cardiac L-type CaV1.2 channel is a multi-subunit protein complex composed of the main pore-forming CaV&#945;1 subunit and CaV&#223; and CaV&#945;2&#948;1 auxiliary subunits7-12. Note that the full complement of auxiliary subunits is required to produce functional CaV1.2 channels at the plasma membrane and dynamic interactions between these subunits are essential to support the normal electric function of the heart13. CaV&#223; promotes the cell surface expression of CaV1.2 channels through a non-covalent nanomolar hydrophobic interaction14. Co-expression of the CaV&#945;2&#948;1 subunit with CaV&#223;-bound CaV&#945;1 stimulates peak current expression (5 to 10-fold) and promotes channel activation at more negative voltages. Gain-of-function mutations of the pore-forming subunit CaV1.2 have been associated with a form of ventricular arrhythmias called the long QT syndrome15 whereas a host of point mutations in the three main subunits forming the L-type CaV1.2 channel have been identified in subjects suffering from arrhythmias of the short QT syndrome form16,17. Ion channels are membrane proteins that can be investigated from a biochemical perspective (protein chemistry) or using electrophysiological tools (current-generating machines) and often using these complementary approaches. Electrophysiology, in particular whole-cell patch-clamping, is a suitable approach to elucidate the function of ion channels15 but cannot resolve modifications in protein trafficking from changes in their biophysical properties. Protein chemistry has, however, often limited use due to the relatively low expression of large membrane proteins relative to smaller soluble proteins. Robust high-throughput methods using fluorescence readout need to be developed in order to specifically address defects in protein biogenesis causing changes in the cell surface expression of ion channels.
Flow cytometry is a biophysical technology employed in cell counting, sorting, biomarker detection, and protein engineering18. When a sample solution of live cells or particles is injected into a flow cytometer, the cells are ordered into a single stream that can be probed by the machine&#39;s detection system (Figure 1). The first flow cytometer instrument produced in 195619 detected only one parameter but modern flow cytometers have multiple lasers and fluorescence detectors that allow the detection of more than 30 fluorescent parameters20,21. Filters and mirrors (emission optics) direct the light scatter or fluorescent light of cells to an electronic network (photodiode and detectors) that convert the light proportionally to its intensity. Digital data are analyzed using specialized software and the primary output is displayed as a dot plot21.
<img alt="Figure 1" src="/files/ftp_upload/54732/54732fig1.jpg" /> 
Figure 1: Biophysical principles of flow cytometry sorting. Single cells are pushed through a nozzle under high pressure within a stream of sheath fluid which moves them across one or more laser interrogation points. The light beam is deflected by the passing cells and the light collected in the forward direction (Forward Scatter, FCS) is sent to a photodiode that converts the light into a signal proportional to the size of the cell. The light is also collected at a 90&#176; angle to the laser path and sent to detectors (also called photomultipliers (PMT)). This light is routed through dichroic mirrors that permit the detection of the side scatter signal (SSC), which reflects the granularity within the cells, and the fluorescent emissions if excited fluorochromes are present in the cell. Three detectors (Green, Yellow, and Red) are represented with different wavelength bandpass filters, allowing the simultaneous detection of different fluorochromes. The different signals are digitized by an external computer and converted into data that will be analyzed to quantify the characteristics of the cells. <a href="http://cloudflare.jove.com/files/ftp_upload/54732/54732fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The high-throughput capacity of flow cytometers was exploited to quantify the relative membrane expression of recombinant wild-type and trafficking-deficient voltage-gated L-type CaV1.2 channels and associated subunits in live cells. cDNA constructs coding for the proteins were doubly tagged to simultaneously carry an extracellular non-fluorescent epitope that can be detected by an impermeable fluorescent conjugated antibody and an intracellular fluorophore that is constitutively fluorescent. Both the extracellular epitope, inserted in an extracellular loop of the protein, and the intracellular fluorophore, inserted after the C-terminus, are translated with the protein. In this series of experiments, the CaV&#945;2&#948;1 protein was engineered to express an extracellular hemagglutinin (HA) epitope (YPYDVPDYA) detected by an impermeable FITC (Fluorescein isothiocyanate)-conjugated anti-HA and mCherry as the intrinsic intracellular fluorophore. To determine the relative cell surface expression level of the mCherry-CaV&#945;2&#948;1 HA-tagged protein, recombinant cells expressing the fusion protein were harvested after transfection, and stained with the FITC-conjugated mouse monoclonal anti-HA epitope tag antibody (Figure 2). FITC is an organic fluorescent compound that is considerably smaller than enzyme reporters and therefore not as likely to interfere with biological function. mCherry- CaV&#945;2&#948;1-HA overexpressed in tsA-201cells, produces a significant 3-log increase in the FITC fluorescence and mCherry fluorescence on two-dimensional plots22. Given that the HA epitope is located in the extracellular portion of the protein, the fluorescence intensity for FITC obtained in the presence of intact cells reflect the relative index of the cell surface expression of HA-tagged protein. The accessibility of the HA epitope in the constructs is systematically validated by measuring the FITC signal after cell permeabilization. This measure also serves to corroborate the normalized total protein expression since the relative fluorescence intensities for FITC estimated in permeabilized cells are qualitatively comparable to the relative fluorescence values for mCherry measured under permeabilized and non-permeabilized conditions22,23. It is important to note that the intrinsic fluorescence spectrum is shifted toward higher values after permeabilization but that the only value being reported is the change in fluorescence intensity as compared to the control construct. Relative changes in the fluorescence intensity for the test constructs are estimated using the &#916;Mean Fluorescence Intensity (&#916;MFI) values for each fluorophore (mCherry or FITC). Experiments are designed to measure the fluorescence intensity of the test construct relative to the fluorescence intensity of the control construct expressed under the same conditions to limit experimental variations in the intrinsic fluorescence of the fluorophore-conjugated antibody. Two membrane proteins were successfully studied using this assay: the pore-forming subunit of the L-type voltage-gated calcium channel CaV1.214,22 and in a different series of experiments, the extracellular auxiliary CaV&#945;2&#948;1 subunit22,23. The following protocol was used to determine the cell surface expression of the CaV&#945;2&#948;1 subunit of the cardiac L-type CaV1.2 channel under control conditions and after mutations affecting the posttranslational modification of the ion channel. Under standardized experimental conditions, the cell surface fluorescence of FITC increases quasi-linearly with the expression of cDNA coding for the mCherry-CaV&#945;2&#948;1-HA proteins (Figure 5 from reference22).
<img alt="Figure 2" src="/files/ftp_upload/54732/54732fig2.jpg" /> 
Figure 2: Schematic representation of total and membrane labeling in the flow cytometry experimental protocol. The scheme outlines some of the main steps necessary to quantify the relative total and cell surface expression of recombinant ion channels by flow cytometry. Cells are transfected with the double-tagged construction mCherry-CaV&#945;2&#948;1-HA in tsA-201 cells (1) and stained before or after permeabilization (2). Multiparameter data are acquired in a flow cytometer (3) for multivariate analysis (4). <a href="http://cloudflare.jove.com/files/ftp_upload/54732/54732fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="763">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td style="width:143px;">New England Biolabs</td>
			<td style="width:123px;">E0554S</td>
			<td style="width:281px;">Can be substitute with QuickChange site-directed mutagenesis Kit (Agilent, #200523).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubes 1,5 mL</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">72-690-001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubes 15 mL&#160;</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">&#160;62-554-002</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable graduated Tranfer Pipets&#160;</td>
			<td style="width:143px;">VWR</td>
			<td style="width:123px;">160001-192&#160;</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100-mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:123px;">430167</td>
			<td style="width:281px;">For standard culture of HEKT cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35-mm culture dish</td>
			<td style="width:143px;">Falcon</td>
			<td style="width:123px;">353001</td>
			<td style="width:281px;">For standard culture of HEKT cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 1 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1251.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 5 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1253.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 10 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1254.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 25ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1285.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dulbecco&#39;s high-glucose medium</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">12100-046</td>
			<td style="width:281px;">Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fetal Bovine Serum, qualified, heat inactivated, US origin</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">16140-071</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">15140-122</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Lipofectamine 2000&#160;</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">11668-019</td>
			<td style="width:281px;">For liposomal transfection. Can be substituted with calcium phosphate transfection.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">31985-070</td>
			<td style="width:281px;">Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Trypsin-EDTA (1X) 0.05%, phenol red</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">25300-062</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL microtubes</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">72.690.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Phosphate Buffered Saline 1X</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">BP661-10</td>
			<td style="width:281px;">Can be &#34;home-made&#34;.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Anti-HA FITC conjugated antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:123px;">H7411</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">IgG1&#8722;FITC Isotype Control antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:123px;">F6397</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="20">
			<td height="61" rowspan="2" style="height:61px;width:216px;">BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:123px;">554714</td>
			<td style="width:281px;">Fixation/Permeabilization. Permeabilization/Wash solution, store at 4 &#176;C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:143px;"></td>
			<td style="width:123px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">49105161</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">15250061</td>
			<td style="width:281px;">To access cell viability.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Refrigerated Microcentrifuge, 5430R</td>
			<td style="width:143px;">Eppendorf&#160;</td>
			<td style="width:123px;">A14H172200</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Forma Steri-Cycle CO2 Incubator</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">370</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laboratory Platform Rocker</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">545034</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water Bath</td>
			<td style="width:143px;">VWR</td>
			<td style="width:123px;">89032-216</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD FACSARIA III&#160;</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:123px;">648282</td>
			<td style="width:281px;">Flow cytometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FlowJo Software v10</td>
			<td style="width:143px;">FlowJo</td>
			<td style="width:123px;">FlowJo v10 Dongle</td>
			<td style="width:281px;">For data analysis.</td>
		</tr>
	</tbody>
</table>

determination of the relative cell surface and total expression of recombinant ion channels using flow cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

This article describes a reliable protocol to quantify total and cell surface of recombinant ion channels expressed in tsA-201cells by a two-color flow cytometry assay. As an example, the relative cell surface and total protein expression was quantified for the CaV&#945;2&#948;1subunit. In order to perform the two-color flow cytometry assay, CaV&#945;2&#948;1 was doubly tagged to express an extracellular non-fluorescent epitope HA that can be detected by an anti-HA F...

Watch this Scientific Journal Video about Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry at JoVE.com

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

The overall goal of this method is to quantify the cell surface expression of membrane proteins using a fluorescent spaced assay. The main advantage of this technique is that flow cytometry assays deliver quantifiable end points on large volumes of live cells in a single experiment. The implications of this technique extend toward a diagnosis and a therapy of cardiac ventricular arrhythmias because these pathologies are often associated with genetic mutations that cause defects in the trafficking of cardiac ion channels.Generally individuals new to this method struggle to identify a suitable extrorsal insertion site for the epitope that does not hurt their protein function and generate a strong fluorescent signal in the presence of the conjugated antibody. Demonstrating this procedure will be Benoite Bourdin, a research assistant from my lab. To begin, doubley tag DNA constructs with mCherry at the intracellular C-terminus.Also, express an extra cellular facing hemmagglutinin epitope, which can be measured using a fluorescence antibody to estimate the cell surface expression of membrane proteins. Next, culture a plate of TSA201 cells using standard techniques until they reach 90%confluence. For each sample prepare the transfection reagents in two tubes.Prepare one 1.5 milliliter tube with four micrograms of DNA and 250 microliters of reduced serum medium, and a second tube with 10 microliters of the liposome-mediated transfection reagent and 250 microliters of serum reduced culture medium. Gently mix the two tubes and incubate them for five minutes at room temperature. Then, gently combine the contents of tube one and tube two and incubate the liposome DNA complexes for 20 minutes at room temperature.Add the complexes to the cultured cells and gently rock the culture dish. Place the dish back at 37 degrees Celsius and under a 5%carbon dioxide atmosphere for 24 hours. 24 hours after the addition of the transfection reagent, remove the medium from the culture dish and carefully wash the cells with 400 microliters of pre-warmed Trypsin-EDTA.Then, add 400 microliters of Trypsin-EDTA and incubate the dish for five minutes to allow cells to detach from the dish. Once detached, stop the enzyme digestion by adding one milliliter of cold culture medium without penicillin or streptomycin to the cells. Gently pipette the medium over the plate four to five times to wash all the cells from the surface.Then, collect the cells in sterile 1.5 milliliter tubes and immediately place them on ice. For the next steps, use ice-cold solution and keep the cells at four degrees to prevent the internalization of the surface epitope. Also, decrease the cells exposure to light in order to limit the photobleaching of the fluorescent signal.Next, centrifuge the tubes to pellet the cells. Then, carefully aspirate and discard the resulting supernatant. Resuspend the pellet to prepare a single cell suspension using one milliliter of PBS.Then, briefly centrifuge the tubes. Pellet and resuspend the cells once more in order to completely remove the culture medium. Next, resuspend the cell pellet in 600 microliters of PBS.Measure the cell concentration and adjust it to at least three million cells per milliliter. Then, divide the cells into new 1.5 milliliter tubes. Be sure to include the appropriate controls to discriminate specific staining from non-specific staining and for extra cellular staining and also for intracellular staining.The isotope control antibody will help to assess the level of background staining and should match the primary antibody, auspicious isotope and fluorophore. It is important to use the control isotope antibody and the fluorescent antibody at the same concentration. To estimate the cell surface expression of membrane proteins, first label just the extra cellular facing HA epitope using a fluorescent light conjugated antibody.Then, add a FITC-conjugated monoclonal anti-HA antibody at five micrograms per milliliter to the tube. Vortex the cells briefly and then incubate them on a rocker platform in the dark. Remove the cells from the dark and add 900 microliters of PBS.Then centrifuge the tube to pellet the cells and aspirate the supernatant. Next, wash the cells by resuspending the pellet in one milliliter of PBS and briefly vortexing the cells. Then, repeat the centrifuge step to again pellet the cells.Repeat this wash step a total of three times to remove any unbound antibody. After the final wash, resuspend the cells in 500 microliters of PBS and transfer the single cell suspension to a five milliliter flow cytometry tube. Keep the cells in the dark at four degrees Celsius until the sample is run.To estimate the intracellular expression of total protein next label the permeabilized cells. This is accomplished using a similar protocol to the extracellular labeling, but with the addition of a saponin based cell permeabilization buffer. Pellet the cells, discard the supernatant and resuspend the cells in 100 microliters of fixation permeabilization solution directly from the prepared stock.Incubate the cells in the dark at four degrees Celsius for 20 minutes. Then, add 100 microliters of freshly prepared permeabilization washing buffer and briefly vortex the cells. Next, pellet the cells and wash them two more times using the permeabilization washing buffer.Then, add the FITC-conjugated monoclonal anti HA antibody at 5 micrograms per milliliter and 100 microliters of the permeabilization washing buffer. Briefly the vortex the cells and incubate the cells in the dark at four degrees Celsius for 30 minutes. Next, add 100 microliters of the permeabilization washing buffer.Pellet the cells and wash them three times in 100 microliters of the same buffer as previously shown. After the final wash, resuspend the cells in 500 microliters of PBS and transfer the single cell suspension into a five milliliter flow cytometry tube. Run the non-permeabilized and permeabilized cells through the flow cytometer on the same day.To begin, launch the flow cytometry analysis software and import the fcs files created during flow cytometry. Click on the first sample and start the gating process in the plot of side scatter versus forward scatter. Next, draw the P1 gate using the ellipse icon around the live cells to eliminate any debris, dead cells or aggregates.Then, draw the tube parameter contour plot of the mCherry versus the FITC fluorescence intensity. Set the P2 gate around the FITC and mCherry positive cells and the P3 gate around the fluorescence negative cell population. Next, select the P2 and P3 gates and click on the add statistics icon in the original workspace window.Click on count and then click on mean. Finally, apply the gates parameters and statistics to all samples probed by the cytometer. This protocol was used to characterize the role of N-glycosylation on total and cell surface expression of CaV Alpha 2 Delta 1 in TSA201 cells.To access the cell surface protein expression HA was measured on non-permeabilized cells that had N-glycosylation sites disrupted at four sites, 16 sites or no site. The FITC levels were strong in the wild type and cells with N-glycosylation cells disrupted at four locations, but only background levels were found in the cells with 16 locations disrupted. For the permeabilized cells, the FITC fluorescence increased for all conditions, indicating that all transfected, HA-tagged, CaV Alpha 2 Delta 1 proteins were stained and detected by the anti HA FITC antibody.In addition, the mCherry fluorescence levels were similar for permeabilized and non-permeabilized cells, signifying that the permeabilization does not alter the relative mCherry signal. Assays conducted after cell permeabilization showed that total protein expression for the cells with 16 locations disrupted was also significantly decreased, whether it was inferred from the FITC fluorescence in permeabilized cells or from the constitutive mCherry fluorescence measured in non-permeabilized and in permeabilized cells. In addition to this procedure, extra physiological recordings can be performed in order to correlate expression and function of ion channels.This technique also paves the way to researchers in the medical field to study the impact of genetic mutations, posttranslational modifications, microRNAs, small GTPases and the role of chaperones in the trafficking of membrane proteins expressed in recombinant or in native cells. After watching this video you should get a good understanding on how to perform DNA transfection in recombinant cells, to stain NTAC and permeabilized cells, as well as to perform data analysis on the samples. Don't forget that working with live cells requires precautions such as working in the helaminal fluid for the transfixion and wearing gloves when handling chemicals.

determination-relative-cell-surface-total-expression-recombinant-ion

Research

JoVE Journal

Biology

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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

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

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

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

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...