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
<ol>
	<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 N-terminus of mCherry between the SacI and SalI sites (Figure 3B)20. 
	NOTE: Appropriate channel function needs to be tested with the control construct using standard electrophysiological methods24.</li>
</ol>
2. Liposome-mediated Transient Transfection (30 min, All Steps are Performed Under Laminar Flow Hood)
<ol>
	<li>Day 1: Plate half a million of tsA-201 cells (or HEKT) in 35 mm culture dishes with 2 mL of Dulbecco&#39;s high-glucose minimum essential medium (DMEM-HG) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (PS) culture medium. Count cells using a standard hemacytometer. Assess cell viability from a fraction of the cell sample using Trypan Blue. Plate enough cells to reach 90% confluence at the time of transfection.</li>
	<li>Day 2: Change culture medium with 2 ml of fresh pre-warmed (37 &#176;C) culture medium without PS.</li>
	<li>For each transfection sample, prepare two 1.5 ml tubes. In tube 1, dilute 4 &#181;g of DNA in 250 &#181;l of reduced serum medium. In tube 2, mix 10 &#181;l of the liposome-mediated transfection reagent with 250 &#181;l serum-reduced culture medium. Mix gently the transfection reagent before use.</li>
	<li>Incubate for 5 min at room temperature.</li>
	<li>Combine the contents of tube 1 and tube 2, mix gently and incubate at least 20 min at room temperature.</li>
	<li>Add the liposomes/DNA complexes to the cultured cells and gently rock the culture dish to mix.</li>
	<li>Incubate at 37 &#176;C under 5% CO2 atmosphere for 24 hr.</li>
</ol>
3. Staining of Cells for Flow Cytometry (3 hr)
<ol>
	<li>Preparing Cell Samples
	<ol>
		<li>Day 3: Remove medium from the culture dish carefully and wash the cells with 400 &#181;l of pre-warmed (37 &#176;C) 0.05% trypsin-1x EDTA (ethylenediaminetetraacetic acid).</li>
		<li>Add 400 &#181;l of trypsin-EDTA and incubate the dish at 37 &#176;C under 5% CO2 atmosphere for 5 min to allow cells to detach from the dish.</li>
		<li>Stop the enzyme digestion by adding 1 ml of cold culture medium without PS and wash out all the cells from the surface by pipetting gently 4-5 times. Avoid over-digestion and over-pipetting to reduce cell death.</li>
		<li>Collect cells in 1.5 ml tubes and place immediately on ice. Use ice cold solutions and keep the cells at 4 &#176;C to prevent the internalization of surface antigens. Decrease lighting to limit photobleaching of the fluorescent signal.</li>
		<li>Centrifuge tubes at 400 x g for 5 min at 4 &#176;C. Carefully aspirate and discard the supernatant.</li>
		<li>Re-suspend the pellet in 1 ml of phosphate-buffered saline 1x (PBS) to prepare a single cell suspension.</li>
		<li>Briefly vortex the tubes very gently and repeat steps 3.1.5 and 3.1.6 to completely remove culture medium.</li>
		<li>Re-suspend the pellet in 600 &#181;l of 1x PBS and adjust the cell concentration to a minimum of 3 x 106 cells/ml.</li>
		<li>Divide the cells in two new 1.5 ml tubes for extracellular and intracellular staining. Include appropriate controls to discriminate specific staining from non-specific staining. 
		NOTE: The isotype control antibody helps assessing the level of background staining and should ideally match each primary antibody&#39;s host species, isotype and fluorophore. Use isotype control and conjugated antibody at the same protein concentration.</li>
	</ol>
	</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/54732/54732table1.jpg" /> 
Table 1: Flow-cytometry experiment control samples for non-permeabilized and permeabilized cells. Each experiment needs to include the following negative controls: (1) Nontransfected cells (without antibody, with the isotype or with the conjugated antibody). (2) Transfected cells with the protein of interest subcloned in a plasmid without constitutive fluorescent intracellular fluorochrome (pCMV- CaV&#945;2&#948;1-HA) or with the doubly tagged construction (pmCherry-CaV&#945;2&#948;1 and incubated without antibody, with the isotype or with the conjugated antibody). Single color controls are used for compensation of fluorochrome emission overlap. The same controls are run for non-permeabilized and permeabilized conditions in each series of experiments.
<ol start="2">
	<li>Cell Surface Staining of Intact Live Cells
	<ol>
		<li>Aliquot 1 x 106 cells/100 &#181;l in 1.5 ml tubes.</li>
		<li>Add the FITC-conjugated monoclonal anti-HA antibody at 5 &#181;g/ml and, vortex before incubating the cells on a rocker platform (200 rpm) in the dark at 4 &#176;C for 45 min. 
		NOTE: The optimal concentration of antibody was determined in preliminary titration experiments (Figure 4).</li>
		<li>Remove the cells from the dark and add 900 &#181;l of 1x PBS/tube. Centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 1 ml of 1x PBS, vortex and centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Repeat the wash (step 3.2.4) twice to remove any unbound antibody. If an unconjugated primary antibody is used, incubate with the appropriate secondary antibody.</li>
		<li>After the final wash, resuspend the cells in 500 &#181;l of 1x PBS and transfer the single cell suspension in 5 ml flow cytometry tubes. Keep the cells in the dark at 4 &#176;C until running the sample.</li>
		<li>Run the samples on a flow cytometer. For best results, analyze the cells on the flow cytometer as soon as possible and no later than 24 hr after.</li>
	</ol>
	</li>
	<li>Intracellular Staining: Fixation, Permeabilization, and Staining
	<ol>
		<li>Aliquot 1 x 106 cells/100 &#181;l in 1.5 ml tubes and centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Discard supernatant and resuspend cells in 100 &#181;l of fixation-permeabilization solution directly from stock.</li>
		<li>Incubate in the dark at 4 &#176;C for 20 min.</li>
		<li>Add 100 &#181;l of freshly prepared 1x permeabilization-washing buffer (dilute 10x permeabilization-washing buffer in distilled H2O). Vortex and sediment cells using a table centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate and discard the supernatant.</li>
		<li>Repeat steps 3.3.4 and 3.3.5.</li>
		<li>Add FITC-conjugated monoclonal anti-HA antibody at 5 &#181;g/ml in 100 &#181;l of 1x permeabilization-washing buffer and, vortex before incubating cells in the dark at 4 &#176;C for 30 min. 
		NOTE : The intracellular staining is performed following the same procedure as the one used for cell surface staining. Saponin-mediated cell permeabilization is however, a quickly reversible process, therefore it is important to replace 1x PBS with 1x Perm/Wash buffer to keep the cells in the constant presence of saponin during intracellular staining.</li>
		<li>Remove the cells from the dark and add 100 &#181;l permeabilization-washing buffer. Centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate carefully the supernatant and resuspend the pellet in 100 &#181;l permeabilization-washing buffer, vortex and centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Repeat the wash (step 3.3.9) one more time to remove any unbound antibody.</li>
		<li>After the final wash, resuspend the cells in 500 &#181;l of 1x PBS and transfer the single cell suspension to 5 mL flow cytometry tubes. Keep the cells in the dark at 4 &#176;C until injecting the sample into the flow cytometer.</li>
		<li>Run the samples on a flow cytometer. Run the fixed samples on the cytometer as soon as possible but no later than 1 week after staining. Run the non-permeabilized and permeabilized cells on the same day.</li>
	</ol>
	</li>
</ol>
4. Flow Cytometry
<ol>
	<li>Flow Cytometer Cell Sorter Daily Setup
	<ol>
		<li>Turn on the flow cytometry software. Prior to experiment, calibrate and setup the flow cytometer cell sorter to ensure optimal instrument performance (i.e. laser and optics are performing to specification, the laser and flow cell are properly aligned) by using instrument setup beads.</li>
		<li>Use the 100 &#181;m nozzle with 20 psi sheath pressure. 
		NOTE: The nozzle does not have to be changed on a bench flow cytometer.</li>
		<li>Set the cytometer&#39;s flow rate according to the manufacturer specification. Exceedingly high flow rates will decrease sensitivity in the detection of variations in fluorescence.</li>
		<li>Select blue (488 nm to excite Fluorescein Isothiocayanate or FITC) and yellow-green (561 nm to excite mCherry) lasers. Collect FITC and mCherry fluorescence levels with a 530/30 nm and with a 610/20 nm bandpass filter respectively.</li>
		<li>Acquire the forward scatter (FCS) versus side scatter (SSC) dot plot for unstained cells using linear scale. Adjust each detector&#39;s amplification to visualize cells in the lower left quadrant of the dot plot.</li>
	</ol>
	</li>
	<li>Sample Reading of Intact Non-permeabilized Cells
	<ol>
		<li>Set the P1gate for live non-permeabilized cells by delineating a free form around the cells to be analyzed excluding cell debris and cell aggregates, thus limiting the fluorescence signal to intact cells. 
		NOTE: Live/dead exclusion dyes can be used to facilitate gate placement on live cells. Set 10,000 events to record in the stopping gate P1. Set this to a higher number of events if need be.</li>
		<li>Acquire mCherry versus FITC two-parameter contour plot to detect baseline autofluorescence of unstained cells. Use bi-logarithmic scale to show negative values and improve resolution between populations25. Adjust each detector&#39;s voltage to set the unstained negative cells within the lower portion of the first ten units of the log fluorescence intensity plots.</li>
		<li>Acquire all intact non-permeabilized samples using settings established in 4.1.5 and 4.1.6 and collect FSC, SSC and signals in the fluorescence detectors.</li>
		<li>Export and save *.fcs files to be used for analysis using flow cytometry analysis software.</li>
	</ol>
	</li>
	<li>Sample Reading of Permeabilized Cells
	<ol>
		<li>Move the P1 gate to select live cells in the permeabilized samples and adjust FSC and SSC voltage as shown in 4.1.5 and 4.1.6.</li>
		<li>Acquire all permeabilized samples and collect FSC, SSC and signals in the fluorescence detectors.</li>
		<li>Export and save *.fcs files to be used for analysis using flow cytometry analysis software.</li>
	</ol>
	</li>
	<li>Data Analysis
	<ol>
		<li>Launch the flow cytometry analysis software and import *.fcs files saved in 4.2.4 and 4.3.3.</li>
		<li>Click on the first sample listed in the workspace window. A new window named after the tube I.D. number opens automatically. Start the gating process in the plot of SSC versus FSC. Draw a gate (P1) using the Ellipse icon around live cells and eliminate any debris, dead cells, or aggregates which have different forward scatter and side scatter than live cells</li>
		<li>To draw the two-parameter contour plot of the mCherry (y-axis) versus FITC (x-axis) fluorescence intensity of the live cells, click first on the x-axis and choose the FITC-A channel and then click on the y-axis and choose the PE-mCherry-A channel. Click on the &#34;Quad&#34; icon to position the quadrant marker at the edge of autofluorescent cells in each fluorescence channel. 
		NOTE: The gate set around the FITC and mCherry positive cells is the P2 gate. The fluorescence negative cell population is referred to as the P3 gate. See Figure 5 for the representative gating method used in this article.</li>
		<li>Select P2 and P3 gates and click on the &#34;Add Statistics&#34; icon in the original workspace window. Click on &#34;Count&#34; (number of positive cells) and click on &#34;Mean&#34; (Mean Fluorescence Intensity of each fluorochrome) or &#34;Median&#34; (Median Fluorescence Intensity of each fluorochrome) statistics among the list of options. Click on the &#34;Add Statistics&#34; icon again. All these values are automatically transferred to the original workspace window. 
		NOTE: The &#34;Mean&#34; is used only if the fluorescence intensity follows a normal distribution. In every other case, click on the &#34;Median&#34; tab. MFI thus could refer to Mean Fluorescence Intensity or Median Fluorescence Intensity. 
		NOTE: The next step is to apply the gates&#39; parameters and statistics to all samples probed by the cytometer.</li>
		<li>In the workspace window, use the mouse to drag and drop the gates and statistics parameters onto the line marked ALL SAMPLES.</li>
		<li>Generate a batch report of two-dimensional contour plots (mCherry vs FITC) and histograms (cell count versus fluorescence intensity) for non-permeabilized and permeabilized cells (Figures 6A-B).</li>
		<li>From the statistics generated in step 4.4.4, calculate the mean fluorescence intensity (MFI) for each fluorochrome for the stained cells. From this value, subtract the MFI value obtained from unstained cells to quantify the surface and total expression of the protein of interest.</li>
		<li>Report the &#916;MFI values for each fluorophore (mCherry and FITC) (Figure 6C-D). Normalize the &#916;MFI measured for the CaV&#945;2&#948;1 construct mutants to the &#916;MFI value obtained for FITC and mCherry with the WT construct. 
		NOTE: The absolute value of fluorescence intensity could vary sharply depending upon the batch of antibodies and the technical abilities of each lab worker, hence the need to normalize fluorescence intensity of the mutant construct using the WT construct.</li>
	</ol>
	</li>
</ol>

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J., Gamper, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Channels: Methods and Protocols. , 233-241 (2013).</li><li>Gonzalez-Gutierrez, G., Miranda-Laferte, E., Neely, A., Hidalgo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Src+Homology+3+Domain+of+the+beta-Subunit+of+Voltage-gated+Calcium+Channels+Promotes+Endocytosis+via+Dynamin+Interaction.">The Src Homology 3 Domain of the beta-Subunit of Voltage-gated Calcium Channels Promotes Endocytosis via Dynamin Interaction.</a> J. Biol.Chem. 282, 2156-2162 (2007).</li><li>Galizzi, J. P., Borsotto, M., Barhanin, J., Fosset, M., Lazdunski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+photoaffinity+labeling+of+receptor+sites+for+the+Calcium+channel+inhibitors+d-cis-diltiazem,+(+/-)-bepridil,+desmethoxyverapamil,+and+(+)-PN+200-110+in+skeletal+muscle+transverse+tubule+membranes.">Characterization and photoaffinity labeling of receptor sites for the Calcium channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes.</a> J. Biol.Chem. 261, 1393-1397 (1986).</li><li>Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+voltage+sensor+in+voltage-dependent+ion+channels.">The voltage sensor in voltage-dependent ion channels.</a> Physiol.Rev. 80, 555-592 (2000).</li><li>Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+variance+of+sodium+current+fluctuations+at+the+node+of+Ranvier.">The variance of sodium current fluctuations at the node of Ranvier.</a> J Physiol. 307, 97-129 (1980).</li><li>Bailey, M. A., Grabe, M., Devor, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+PCMBS-dependent+modification+of+KCa3.1+channel+gating.">Characterization of the PCMBS-dependent modification of KCa3.1 channel gating.</a> J. Gen. Physiol. 136, 367-387 (2010).</li><li>Fletcher, P. A., Scriven, D. R., Schulson, M. N., Moore, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Image+Colocalization+and+Its+Statistical+Significance.">Multi-Image Colocalization and Its Statistical Significance.</a> Biophys. J. 99, 1996-2005 (2010).</li><li>Lizotte, E., Tremblay, A., Allen, B. G., Fiset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+subcellular+protein+fractions+from+mouse+heart.">Isolation and characterization of subcellular protein fractions from mouse heart.</a> Anal. Biochem. 345, 47-54 (2005).</li><li>Mattheyses, A. L., Simon, S. M., Rappoport, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+with+total+internal+reflection+fluorescence+microscopy+for+the+cell+biologist.">Imaging with total internal reflection fluorescence microscopy for the cell biologist.</a> J. Cell Sci. 123, 3621-3628 (2010).</li><li>Yamamura, H., Suzuki, Y., Imaizumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+light+on+ion+channel+imaging+by+total+internal+reflection+fluorescence+(TIRF)+microscopy.">New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.</a> J. Pharmacol. Sci. 128, 1-7 (2015).</li><li>Wible, B. A., 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=HERG-Lite-R:+A+novel+comprehensive+high-throughput+screen+for+drug-induced+hERG+risk.">HERG-Lite-R: A novel comprehensive high-throughput screen for drug-induced hERG risk.</a> J. Pharmacol. Toxicol. Methods. 52, 136-145 (2005).</li><li>Wilde, A. A. M., Brugada, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypical+Manifestations+of+Mutations+in+the+Genes+Encoding+Subunits+of+the+Cardiac+Sodium+Channel.">Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Sodium Channel.</a> Circ. Res. 108, 884-887 (2011).</li><li>Milano, A., 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=Sudden+Cardiac+Arrest+and+Rare+Genetic+Variants+in+the+Community.">Sudden Cardiac Arrest and Rare Genetic Variants in the Community.</a> Circ Cardiovasc Genet. , (2016).</li><li>Schnell, U., Dijk, F., Sjollema, K. A., Giepmans, B. N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolabeling+artifacts+and+the+need+for+live-cell+imaging.">Immunolabeling artifacts and the need for live-cell imaging.</a> Nat. Meth. 9, 152-158 (2012).</li></ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>Q5 Site-Directed Mutagenesis Kit</td><td>New England Biolabs</td><td>E0554S</td><td>Can be substitute with QuickChange site-directed mutagenesis Kit (Agilent, #200523).</td></tr><tr><td>Tubes 1.5 ml</td><td>Sarstedt</td><td>72-690-001</td><td></td></tr><tr><td>Tubes 15 ml&amp;nbsp;</td><td>Sarstedt</td><td>&amp;nbsp;62-554-002</td><td></td></tr><tr><td>Disposable graduated Tranfer Pipets&amp;nbsp;</td><td>VWR</td><td>160001-192&amp;nbsp;</td><td></td></tr><tr><td>100 mm culture dish</td><td>Corning</td><td>430167</td><td>For standard culture of HEKT cells.</td></tr><tr><td>35 mm culture dish</td><td>Falcon</td><td>353001</td><td>For standard culture of HEKT cells.</td></tr><tr><td>Serological pipette 1 ml</td><td>Sarstedt</td><td>86.1251.001</td><td></td></tr><tr><td>Serological pipette 5 ml</td><td>Sarstedt</td><td>86.1253.001</td><td></td></tr><tr><td>Serological pipette 10 ml</td><td>Sarstedt</td><td>86.1254.001</td><td></td></tr><tr><td>Serological pipette 25 ml</td><td>Sarstedt</td><td>86.1285.001</td><td></td></tr><tr><td>Dulbecco&#039;s high-glucose medium</td><td>Life Technologies</td><td>12100-046</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated, US origin</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Lipofectamine 2000&amp;nbsp;</td><td>Life Technologies</td><td>11668-019</td><td>For liposomal transfection. Can be substituted with calcium phosphate transfection.</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-070</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Trypsin-EDTA (1x) 0.05%, phenol red</td><td>Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>1.5 ml microtubes</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>&amp;nbsp;Phosphate Buffered Saline 1x</td><td>Fisher</td><td>BP661-10</td><td>Can be &quot;home-made&quot;.</td></tr><tr><td>Anti-HA FITC conjugated antibody</td><td>Sigma</td><td>H7411</td><td></td></tr><tr><td>IgG1&amp;minus;FITC Isotype Control antibody</td><td>Sigma</td><td>F6397</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td><td>BD Biosciences</td><td>554714</td><td>Fixation/Permeabilization. Permeabilization/Wash solution, store at 4 &amp;deg;C.</td></tr><tr><td>Hemacytometer</td><td>Fisher</td><td>49105161</td><td></td></tr><tr><td>Trypan Blue</td><td>Fisher</td><td>15250061</td><td>To access cell viability.</td></tr><tr><td>Refrigerated Microcentrifuge, 5430R</td><td>Eppendorf&amp;nbsp;</td><td>A14H172200</td><td></td></tr><tr><td>Forma Steri-Cycle CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher</td><td>370</td><td></td></tr><tr><td>Laboratory Platform Rocker</td><td>Fisher</td><td>545034</td><td></td></tr><tr><td>Water Bath</td><td>VWR</td><td>89032-216</td><td></td></tr><tr><td>BD FACSARIA III&amp;nbsp;</td><td>BD Biosciences</td><td>648282</td><td>Flow cytometer.</td></tr><tr><td>FlowJo Software v10</td><td>FlowJo</td><td>FlowJo v10 Dongle</td><td>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 ...

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

Research

JoVE Journal

Biology

13.2K Views.  Montreal Heart Institute Research Centre.  Erfelijke hartritmestoornissen worden vaak veroorzaakt door mutaties die de oppervlakte levering van één of meer ionkanalen wijzigen. Hier passen we flowcytometrie assays een kwantificering van de relatieve totale en celoppervlak-eiwit expressie van recombinant ionenkanalen tot expressie gebracht in TSA-201 cellen. 

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

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

Genetics

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Cancer Research

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

Biochemistry

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) ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Immunology and Infection

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

A Flow Cytometry Protocol for Understanding Export Mechanisms

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, microRNAs, and other noncoding RNAs), from one cell to another, leading to phenotypic consequences in the recipient cells. Of all the various EV cargo, microRNAs (miRNAs) have garnered a great deal of attention for their role in shaping the microenvironment and in educating recipient cells because of their clear dysregulation and abundance in EVs. Additional data indicates that many miRNAs are actively loaded into EVs. Despite this clear evidence, research on the dynamics of export and mechanisms of miRNA sorting is limited. Here, we provide a protocol using flow cytometry analysis of EV-miRNA that can be used to understand the dynamics of EV-miRNA loading and identify the machinery involved in miRNA export. In this protocol, miRNAs predetermined to be enriched in EVs and depleted from donor cells are conjugated to a fluorophore and transfected into the donor cells. The fluorescently tagged miRNAs are then verified for loading into EVs and depletion from cells using qRT-PCR. As both a transfection control and a tool for gating the transfected population of cells, a fluorescently labeled cellular RNA (cell-retained and EV-depleted) is included. Cells transfected with both the EV-miRNA and cell-retained-miRNA are evaluated for fluorescent signals over the course of 72 h. The fluorescence signal intensity specific for the EV-miRNAs diminishes rapidly compared to the cell-retained miRNA. Using this straightforward protocol, one could now assess the dynamics of miRNA loading and identify various factors responsible for loading miRNAs into EVs.

Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression 

Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...

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

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