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.

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