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>

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	<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...

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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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.2K Views.  Montreal Heart Institute Research Centre. 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.

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

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 ...

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 ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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 ...

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

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Chapters in this video

Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

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

Flow Cytometry Purification of Mouse Meiotic Cells

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

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

Flow Cytometry-based Purification of S. cerevisiae Zygotes

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

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

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry