This work was supported by ERC starting grants (ERC-2011-StG 280255 and ERC-2013-StG 337479) and by the Netherlands Organization for Health Research and Development (Vidi 91713319).

1. Transient Transfection
Note: CytoBAS and NucleoBAS (Please see Materials Table) are successfully used in several cell types, (U2OS, Huh7, HepG2, H69, MDCK and HEK293T cells). The main requirement to use the sensor is that it needs to be expressed, requiring the encoding DNA to enter the cell.
<ol>
	<li>Harvest cells from an 80% confluent 25 cm2 flask. Dilute cells in complete culture medium suitable for the specific cell line (10% FBS, 1% L-glutamine, 1% pen/strep for U2OS and Huh7 cells).</li>
	<li>Plate cells at the desired density into a sterile 8 well chambered cover glass (0.8 cm2). Aim for a sub-confluent (60-80% confluency) layer of cells on the day of the experiment, although this is not essential.</li>
	<li>Add complete culture medium to a final volume of 400 &#181;l per well. Allow cells to attach for 24 hr.</li>
	<li>Prepare the transfection mix in a sterile tube by adding 0.5 &#181;g of NucleoBAS or CytoBAS DNA to 100 &#181;l of culture medium (serum/antibiotic free). Vortex well. Add a transfection reagent and mix by vortexing. Transfection reagents that give optimal efficiency are cell type dependent and might require prior testing.
	<ol>
		<li>For instance, use 2.5 &#181;l of 1 mg/ml polyethylenimine (PEI) and incubate the DNA/PEI mix for 15 min at RT.</li>
	</ol>
	</li>
	<li>Add the transfection mixture in droplets to the cells and mix by gently shaking the well plate by hand. Add 100 &#181;l of the mixture for a well of ~9.6 cm2. Use 10 &#181;l of the transfection mixture for individual imaging chambers for transiently transfected cells. 
	Note: After 24-48 hr, cells will express the bile acid sensor and can be used for experiments.</li>
</ol>
2. Stable Transfection
<ol>
	<li>Harvest cells from an 80% confluent 25 cm2 flask. Dilute pellet in complete culture medium suitable for the specific cell line (10% Fetal Bovine Serum, 1% L-glutamine, 1% pen/strep for U2OS and Huh7 cells) to a concentration of 150,000 cells per ml and add around 300,000 cells per well in a 6-well culture plate (2 ml end volume).</li>
	<li>Allow cells to attach for 24 hr.</li>
	<li>Prepare the transfection mix in a sterile tube by adding 0.5 &#181;g of CytoBAS or NucleoBAS DNA (Please see Materials Table) to 100 &#181;l of culture medium (serum/antibiotic free). Vortex well. Add a transfection reagent and mix by vortexing.
	<ol>
		<li>For instance, use 2.5 &#181;l of 1 mg/ml polyethylenimine (PEI) and incubate the DNA/PEI mix for 15 min at RT.</li>
	</ol>
	</li>
	<li>Add 100 &#181;l of the transfection mixture in droplets to the cells and mix by gently shaking the well plate by hand. Always include an untransfected control when creating a stable cell line.</li>
	<li>Grow cells O/N at 37 &#176;C, 5% CO2.</li>
	<li>Trypsinize cells according to manufacturer&#39;s protocol (Incubate cells at 37 &#176;C with 1.5 ml prewarmed trypsin per 25 cm2 cell culture) and spin them down at 250 x g at RT&#160;for 5 min. Remove supernatant and resuspend cells in 13 ml of complete culture medium. Divide cells over various 10 cm diameter culture dishes: 0.5 ml (low density), 1.5 ml (medium density), and 4.5 ml (high density). Do the same for remaining 6.5 ml.</li>
	<li>Add culture medium to a final volume of 10 ml. For U2OS cells, use 800 &#181;g/ml G418 for plasmids with the neomycin resistance cassette such as the CytoBAS and NucleoBAS constructs to select for positive cells. This concentrations is cell type dependent and can be determined by selecting the lowest antibiotic (G418) concentration where untransfected cells died within 2 to 10 days (800 &#181;g/ml G418 for U2OS cells).</li>
	<li>Refresh the culture medium with the appropriate antibiotic (G418) every 72 hr until the untransfected cells are dead.</li>
	<li>Examine the culture plate under the microscope with a 20X objective for positive colonies (fluorescence can be monitored using most filter sets used for green, cyan or yellow fluorescence).</li>
	<li>Mark the positive colonies that lie isolated from other colonies with a pen on the exterior bottom of the culture dish.</li>
	<li>Wash the plate twice with Phosphate Buffered Saline (PBS) and aspirate it. Take around 15 sterile cloning cylinders and dip them into sterile silicone. Apply them around the selected colonies by pressing lightly against the petri-dish and make sure that not more than one colony is included in the cloning ring.</li>
	<li>Pipet 20 &#181;l of pre-warmed trypsin (1x, 0.25%) in the cloning cylinder and incubate at 37 &#176;C until most of the cells have rounded up (observe with a microscope).</li>
	<li>Add 150 &#181;l of culture medium with serum and the appropriate antibiotic (10% FBS and 800 &#181;g/ml G418 for U2OS and Huh7 cells) in the cloning cylinder and pipet up and down several times to resuspend the cells and transfer to a 96-well plate. Refresh medium after 4-24 hr and allow cells to grow confluent in the next few days (37 &#176;C, 5% CO2for U2OS and Huh7 cells).</li>
	<li>Refresh medium with antibiotic every 3 or 4 days. When the cells have grown confluent, transfer them to a larger well plate. Repeat this step until the culture is large enough for experiments. Cryopreserve some vials for backup. Cryopreservation medium consists of 20% FBS and 20% DMSO in media (DMEM) without supplements. 
	Note: Now, the cell line is monoclonal and considered stable for expressing the Bile Acid Sensor. Half the concentration of the antibiotic G418 (400 &#181;g/ml) is now sufficient to maintain expression in the stable cell line.</li>
</ol>
3. Lentiviral Transduction
Note: Some cell lines are considered difficult to transfect by more traditional methods such as the polyethylenimine (PEI) method. Viral transduction of cells is an efficient alternative tool for gene-delivery and stable transgene expression.
<ol>
	<li>Harvest cells from an 80% confluent 25 cm2 flask. Dilute cells in complete culture medium suitable for the specific cell line (10% FBS, 1% L-glutamine, 1% pen/strep for U2OS and Huh7 cells) and add around 200,000 cells per well in a 6-well culture plate.</li>
	<li>Add culture medium to a final volume of 2 ml per well. Allow cells to attach for 24 hr.</li>
	<li>Perform viral transductions by incubating cells for 4-6 hr with 500 &#181;l CytoBAS lentivirus containing medium (available upon request) filled up to a total amount of 1 ml with complete culture medium containing 10 &#181;g/ml diethylaminoethyl (DEAE)-dextran or another lentiviral transduction enhancer. Do not forget to include a well with untransduced control cells.</li>
	<li>Remove medium and add 2 ml of complete culture medium containing the desired antibiotic (500 &#181;g/ml hygromycin). Use a lentiviral construct for CytoBAS transduction that provides hygromycin resistance to cells. Grow cells under desired conditions.</li>
	<li>Refresh medium with (500 &#181;g/ml hygromycin) antibiotic every 3 days and split the cells when 80% confluent, until all negative control cells are dead (takes about 1-2 weeks for most cell lines). The cell line is now considered stable for expressing the Bile Acid Sensor.
	<ol>
		<li>Alternatively, use cells for experiments 1-3 days after viral transduction. As live virus might be present, this requires FRET-readout equipment in rooms with the appropriate safety level.</li>
	</ol>
	</li>
	<li>Alternatively, for rapid isolation of stable cell lines, perform fluorescence activated cell sorting (FACS).
	<ol>
		<li>Harvest cells from a confluent 160 cm2 flask.</li>
		<li>Dilute cells in Leibovitz culture medium (L-15 medium) with &#60;5% serum to a concentration of maximum 5 x 106 cells per ml</li>
		<li>For collection of sorted cells, fill FACS tubes with 1 ml of collection medium (L-15 medium + 1.5% pen/strep, 1% L-glut and 20% serum).</li>
		<li>Sort cells for citrine (520-580 nm) or cerulean (450-520 nm), excited with 405 laser.</li>
		<li>After sorting approximately 500,000 cells, spin down cells (5 min, 250 x g) and resuspend them in 5 ml normal complete culture medium (10% FBS, 250 &#181;g/ml Hygromycin for U2OS and Huh7 CytoBAS cells) and plate them in a T25 culture flask. Grow cells under desired conditions (37 &#176;C, 5% CO2 for U2OS and Huh7 cells).</li>
	</ol>
	</li>
</ol>
4. Live Cell Imaging of the Bile Acid Sensor
Note: Cells containing bile acid transporters can be cultured in medium with 1-10% charcoal-filtered serum that removes lipophilic compounds. Normal serum often contains bile acids that could lead to intracellular bile acid accumulation and saturation of the Bile Acid Sensor.
<ol>
	<li>FRET measurements using the confocal microscope
	<ol>
		<li>Plate the cells stably or transiently expressing the sensor at the desired density into a sterile 8 well coverslip bottomed chamber slide (0.8 cm2). Grow cells in complete culture medium (using charcoal-filtered serum) so that on the day of the experiment, confluency is around 60-70%.</li>
		<li>Wash the adherent cells in chamber slide once with 200 &#181;l 1x PBS or Leibovitz&#39;s L-15 culture medium.</li>
		<li>Aspirate and replace with 300 &#181;l Leibovitz&#39;s L-15 culture medium so that no CO2control is necessary. 
		Note: DMEM without phenol red can be used as well but requires CO2buffered conditions. The bile acid sensor shows (some) pH-sensitivity so aim to keep the pH constant during the data-collection.</li>
		<li>Dilute the compounds to be used in the confocal imaging experiment also in L-15 culture medium. Take into account that during imaging, relatively large amounts of liquid in the chambers have to be added to secure rapid mixing of the sample (50-100 &#181;l), so make 3-5x solutions.</li>
		<li>Start the imaging software of a confocal microscope with a 37 &#176;C incubation chamber and turn on the violet 405 nm laser. Put a drop of immersion oil onto the objective and place the 8-chamber on top of it. For single cell imaging of FRET, use the 63X oil objective. The sensor has been successfully used at RT, but cell behavior might be altered.</li>
		<li>Set the settings of the confocal microscope properly.
		<ol>
			<li>Set Acquisition mode: xyt (time-lapse imaging of single focal plane).</li>
			<li>Acquire images at 20 sec intervals to allow compounds to be added between acquisitions without pausing the experiment.</li>
			<li>Set spectral range for emission detection: Cerulean: 450-520 nm; Citrine: 520-580 nm.</li>
		</ol>
		</li>
		<li>Focus on the single cell layer using transillumination light. Start imaging and adjust the z-position more precisely. Adjust gain and offset for each channel to distinguish signal from background while remaining well below saturation for all pixels in cells of interest.</li>
		<li>Draw circles to define the region of interest (ROI). Select cells that show similar fluorescence intensity. Avoid cells that obviously differ from the average cell in size and shape. Expose the cells to light for a time as short as possible to minimize photobleaching of the sensor. It is advisable to draw ROIs not very close to the cell perimeter. This area is most sensitive to changes in fluorescence intensity due to focal drift (cells moving in z-direction) making it more difficult to monitor changes in fluorescence ratio during the experiment.</li>
		<li>Start measurements with the confocal microscope. Wait until the cerulean and citrine fluorescence is stable.</li>
		<li>Add 50-100 &#181;l bile acids or other compounds at chosen time points during measuring. The relatively high amounts of liquid (one-fifth to one-third of the final volume) are necessary to mix the fluids well (within 10 sec) without shaking/pipetting up and down. Make sure not to touch the edge of the 8-well chamber with the pipette tip and add the liquid slowly so that the cells do not get out of focus. Do not add new compounds before the plateau phase is reached. This takes about 200 sec.</li>
		<li>End each experiment with the addition of 100 &#181;l GW4064, end concentration 5-10 &#181;M (sensor saturating dose) and wait until sensor fluorescence is stable.</li>
		<li>Save the experiment and export the data as an .AVI file.</li>
		<li>Open in ImageJ both AVI files (channel 00, cerulean; channel 01, citrine) by selecting Plugins &#62; Stacks &#62; Stack interleaver. Alternatively, import confocal file (e.g., .lsm or .lif files) directly in Image J using appropriate plugins (available at http://www.openmicroscopy.org) and move to step 4.1.14.
		<ol>
			<li>For Stack 1: use Channel 01 (citrine).</li>
			<li>For Stack 2: use Channel 00 (cerulean).</li>
		</ol>
		</li>
		<li>Click on Edit &#62; Selection &#62; Add to manager, to open the ROI manager window. Check the checkbox &#39;Show All&#39;.</li>
		<li>Draw a few regions of interest (ROIs) covering specific cells with the oval selection tool. Also draw one circle in an area outside cells or inside a cell that does not express the sensor to determine the background signal. It is advisable to draw ROIs not very close to the cell perimeter in experiments when changes in fluorescence intensity due to focal drift (cells moving in z-direction) or cell migration were obvious.</li>
		<li>Select one cell. Click on Plugins &#62; Ratio Profiler. This will result in 3 screens: RAW, ratio and Ratio_Profile. The RAW window shows the increase in intensity of citrine (blue line) and a decrease in cerulean (red line) if there is FRET. The Ratio window gives information about the ratio citrine/cerulean, which will increase with an increase in FRET. The Ratio_Profile window gives the actual numbers of fluorescence intensity measured in both channels. If microscope setup-specific files (e.g., .lsm or .lif instead of .avi) files are used the channel order might be reversed.</li>
		<li>Copy the data from the Ratio_Profile window in the spreadsheet attached as supplementary data. Do the same for all the other cells (and background ROI). 
		Note: In the online spreadsheet, all data is normalized to the condition at which maximum BAS activation is expected. Given that GW4064 is the most potent activator of FXR, the fluorescence ratio after incubation with a surplus of GW4064 is set to 1. It is therefore important to end all of the experiments with addition of GW4064. The advantage of this is that the data is no longer dependent on laser intensity or detector gain and experiments on different days can be compared more easily. Furthermore, in the bottom graph of the spreadsheet, a running average can be used to smooth the curves for experimental noise. However, do not use this graph when analyzing kinetic data, since the running average will also smooth fast kinetic responses.</li>
	</ol>
	</li>
	<li>FRET measurements using Fluorescence Activated Cell Sorting (FACS)
	<ol>
		<li>Dilute all compounds for the FACS experiment in sterile FACS uptake buffer (0.3 mM EDTA, 0.5% BSA, 0.01% NaN3and 10 mM D-glucose).</li>
		<li>Harvest cells from an 80% confluent T-160 cm2 cell culture flask using 5 mM EDTA in PBS. Centrifuge cells at 250 x g for 5 min. Wash cell pellet 2x in 5 ml FACS uptake buffer at RT.</li>
		<li>Count cells using the coulter counter or a counting chamber.</li>
		<li>Dilute pellet in FACS uptake buffer to a concentration of 1 x 106 cells/ml. Pipette up and down to create a homogeneous suspension of single cells. If cells are difficult to disaggregate, put the samples through a cell strainer before sorting to minimize nozzle clogs.</li>
		<li>Pipet 200 &#181;l cells per FACS tube and protect them from light.</li>
		<li>Add the desired concentration of the compound (e.g., bile acids, synthetic FXR ligands, transporter inhibitors). Vortex. Incubate for 20-30 min at RT while shaking (in the dark).</li>
		<li>Meanwhile, start the FACS (the lasers need time to warm up).</li>
		<li>Set the flow cytometry gating parameters for the experiment (see Figure 3):
		<ol>
			<li>Load around 100,000-200,000 NucleoBAS or CytoBAS transfected cells to determine the gates.</li>
			<li>First adjust the FSC and SSC voltages to plot the cells in the center of the plot.</li>
			<li>Using the violet laser, adjust the cerulean (450/40 nm) voltage value and citrine (525/20 nm) voltage and ensure that all NucleoBAS or CytoBAS positive cells are plotted within the scatter plot.</li>
			<li>Set the correct gates (Gate P1 up to Gate P4), see Figure 4.
			<ol>
				<li>Use gate P1 to exclude dead cells, usually displayed in the lower left corner by selecting the main population in the middle of the SSC-A/FSC-A plot.</li>
				<li>Use gate P2 in the FSC-H/FSC-A window to remove duplets from analysis. Single cells are presented in a more diagonal line than doublets.</li>
				<li>Gate P3 in the citrine (525/20 nm)/cerulean (450/40 nm) window can be used to select for NucleoBAS/CytoBAS positive cells, by gating citrine and cerulean high cells, a population displayed diagonally in the upper right corner of the plot.</li>
				<li>Draw gate P4 at the top left of the population, as close as possible and make sure that no more than 5% of the cells fall within this gate.</li>
			</ol>
			</li>
			<li>Load some untransfected cells (no expression of CytoBAS or NucleoBAS) to determine auto-fluorescence. The untransfected population may not be located in gate P3. Adjust gate P3 when necessary to exclude auto-fluorescent cells.</li>
		</ol>
		</li>
		<li>Vortex the samples before placing the tubes in the FACS. For each sample, measure at least 10,000 cells in gate P3. 
		Note: The Population Hierarchy Window gives the number of events being displayed for each gate and % of the parent gate. In gate 4, the % of the parent states cells with an increased citrine/cerulean ratio as a percentage of all NucleoBAS positive cells.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Aoki, K., Kamioka, Y., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+imaging+of+cell+signaling+from+in+vitro+to+in+vivo:+basis+of+biosensor+construction,+live+imaging,+and+image+processing.">Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo: basis of biosensor construction, live imaging, and image processing.</a> Dev. Growth Differ. 55 (4), 515-522 (2013).</li><li>Jares-Erijman, E. A., Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+imaging.">FRET imaging.</a> Nature Biotechnol. 21 (11), 1387-1395 (2003).</li><li>Mank, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophys. J. 90 (5), 1790-1796 (2006).</li><li>Li, I. T., Pham, E., Truong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosensors+based+on+the+principle+of+fluorescence+resonance+energy+transfer+for+monitoring+cellular+dynamics.">Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics.</a> J. Biotechnol. Lett. 28 (24), 1971-1982 (2006).</li><li>Stephens, D. J., Allan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+techniques+for+live+cell+imaging.">Light microscopy techniques for live cell imaging.</a> Science. 300 (5616), 82-86 (2003).</li><li>Merkx, M., Golynskiy, M. V., Lindenburg, L. H., Vinkenborg, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+FRET+sensor+proteins+based+on+mutually+exclusive+domain+interactions.">Rational design of FRET sensor proteins based on mutually exclusive domain interactions.</a> Biochem. Soc. Trans. 41 (5), 1201-1205 (2013).</li><li>Lindenburg, L. H., 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=Quantifying+Stickiness:+Thermodynamic+Characterization+of+Intramolecular+Domain+Interactions+To+Guide+the+Design+of+Fo&#776;rster+Resonance+Energy+Transfer+Sensors.">Quantifying Stickiness: Thermodynamic Characterization of Intramolecular Domain Interactions To Guide the Design of Fo&#776;rster Resonance Energy Transfer Sensors.</a> Biochem. 53 (40), 6370-6381 (2014).</li><li>van der Velden, L. M., 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=Monitoring+bile+acid+transport+in+single+living+cells+using+a+genetically+encoded+F&#246;rster+resonance+energy+transfer+sensor.">Monitoring bile acid transport in single living cells using a genetically encoded F&#246;rster resonance energy transfer sensor.</a> J. Hepatol. 57 (2), 740-752 (2013).</li><li>Dawson, P. 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=The+heteromeric+organic+solute+transporter+&#945;-&#946;,+Ost&#945;-Ost&#946;,+is+an+ileal+basolateral+bile+acid+transporter.">The heteromeric organic solute transporter &#945;-&#946;, Ost&#945;-Ost&#946;, is an ileal basolateral bile acid transporter.</a> J. Biol. Chem. 280 (8), 6960-6968 (2005).</li><li>Meier, P. J., Stieger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salt+transporters.">Bile salt transporters.</a> Annu. Rev. Physiol. 64 (1), 635-661 (2002).</li><li>Klarenbeek, J. B., Goedhart, J., Hink, M. A., Gadella, T. W., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mTurquoise-based+cAMP+sensor+for+both+FLIM+and+ratiometric+read-out+has+improved+dynamic+range.">A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.</a> PLoS One. 6 (4), e19170 (2011).</li><li>Wallrabe, H., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+molecules+using+FRET+and+FLIM+microscopy.">Imaging protein molecules using FRET and FLIM microscopy.</a> Curr. Opin Biotechnol. 16 (1), 19-27 (2005).</li><li>Plass, J. R., 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=Farnesoid+X+receptor+and+bile+salts+are+involved+in+transcriptional+regulation+of+the+gene+encoding+the+human+bile+salt+export+pump.">Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump.</a> J. Hepatol. 35 (3), 589-596 (2002).</li></ol>

The authors have nothing to disclose.

Here we present a detailed protocol for the use of a novel genetically encoded bile acid sensor capable of monitoring the spatiotemporal dynamics of bile acid transport in living cells. This biosensor consists of cerulean and citrine fluorescent proteins that are fused to FXR-LBD, thereby forming a FRET-based bile acid sensor (BAS).
The Bile Acid Sensor is relatively simple and convenient in use when having basic experience with cell culture and FACS or (confocal) microscopy. However, some asp...

F&#246;rster Resonance Energy Transfer (FRET) is widely used to gain a better understanding of cellular functions in living cells with high temporal and spatial resolution1. In FRET, energy from an excited donor fluorophore is transferred to an acceptor fluorophore. FRET efficiency is strongly dependent on the distance between the donor and acceptor fluorophore and their orientation and is therefore a sensitive readout of conformational changes that affect the two fluorophores. This phenomenon is exploited to generate FRET-based biosensors for the imaging of small molecules. Changes in their concentration can be monitored as increases/decreases in the ratio of emission intensity of the acceptor versus the donor fluorophore2. For instance, FRET-based calcium biosensors allow for fast and stable detection of free calcium concentrations in living cells3. Other advantages of FRET-based biosensors are imaging in single living cells, their non-invasiveness, their ability to be targeted to different cell types and cellular compartments4.
Many aspects of intracellular bile acid dynamics are still poorly understood. For example, little is known about the mechanism underlying regulation of conjugated and unconjugated bile acid transport. Existing techniques to monitor this transport primarily make use of luciferase-based reporters, radiolabeled bile acids, or fluorescent bile acid analogs. The latter requires modification of bile acids, possibly affecting their properties. Luciferase-based reporters have poor time resolution. Besides, these techniques result in loss of the sample and are not applicable for imaging in single cells. Therefore, it would be beneficial to use methods that allow live single cell imaging of transport activity using FRET biosensors, especially since it includes the advantage of ratiometric detection5,6. While variants of CFP/YFP form most frequently used FRET pairs, new strategies using mOrange and mCherry carrying self-association-inducing mutations have led to an expansion of the FRET toolbox with novel sensors, including a red-shifted bile acid sensor7.
We previously created a genetically-encoded FRET bile acid sensor (BAS), that consists of a donor fluorophore (cerulean) and an acceptor fluorophore (citrine) that are fused with the farnesoid X receptor (FXR) ligand binding domain (FXR-LBD) and a peptide containing an LXXLL motif8. This peptide associates with the FXR-LBD in a bile acid-dependent manner. Upon FXR activation, the distance between citrine and cerulean will alter due to a conformational change. In mammalian cell lines, FXR activation results in a clearly detectable increase in the citrine/cerulean ratio, while the purified sensor works in the opposite direction and leads to a decreased FRET ratio upon FXR activation. This sensor (CytoBAS) allows monitoring of cytosolic bile acid dynamics. By carboxyl-terminal addition of subcellular targeting motifs, the BAS construct can be targeted to the nucleus (NucleoBAS) and peroxisomes (PeroxiBAS), allowing measurements of bile acid concentrations in different cellular compartments. Although the addition of the peroxisomal targeting motif does not impair its responsiveness to bile acids, cell permeable FXR-ligands did not induce any FRET changes of PeroxiBAS inside peroxisomes8. As the nature of this discrepancy is unknown, the protocol below is focused on CytoBAS and NucleoBAS.
The use of this genetically encoded FRET sensor was recently demonstrated in cells containing the hepatic bile acid transporters Na+/taurocholate co-transporting polypeptide (NTCP) and organic solute transporter alpha / beta (OST&#945;&#946;)8. NTCP is the principal hepatic bile acid importer and OST&#945;&#946; is a basolateral intestinal bile acid transporter that can function both as an importer and exporter dependent on the electrochemical bile acid concentration gradient9,10. Recent data showed that upon bile acid transport by NTCP and/or OST&#945;&#946;, robust and fast responses in FRET ratio as a result of ligand-FXR-LBD interaction can be observed.
Here, we describe detailed protocols for methods to measure FRET such as confocal microscopic analysis and fluorescence activated cell sorting (FACS), highlight critical steps, address potential problems and discuss alternative methods. Using this genetically encoded FRET sensor, bile acid interaction with FXR-LBD can be quantified and monitored directly in living cells and provides a rapid and simple method of visualizing bile acid transport and dynamics in real-time. Mammalian expression plasmids encoding CytoBAS and NucleoBAS are available commercially. Therefore, this biosensor can further contribute to the understanding of bile acid transporters or compounds that activate FXR and provide a deeper insight into bile acid biology and signaling.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">CytoBAS&#160;</td>
			<td style="width:173px;">Addgene</td>
			<td align="right" style="width:145px;">62860</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucleoBAS&#160;</td>
			<td style="width:173px;">Addgene</td>
			<td align="right">62861</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Dulbecco&#39;s modified Eagles media (DMEM)</td>
			<td style="width:173px;">Lonza</td>
			<td>BE12-614F</td>
			<td>High glucose without L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin (pen/strep)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">17-602E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">L-glutamine (200mM)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">102-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Trypsin-EDTA (10x)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">CC-5012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T-25 cell culture flask</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0253</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T-175 cell culture flask</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0238</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">6-well plate</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">734-0229</td>
			<td>Poly-L-lysine and Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">10cm dish</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0243</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Diethylaminoethyl (DEAE) - Dextran</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">D9885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Polyethylenimine (PEI)&#160;</td>
			<td style="width:173px;">Brunschwig</td>
			<td style="width:145px;">23966-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">G418 (geneticin) 50 mg/ml</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">10131-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Hygromycin B, 50 mg / ml</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">10687-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cloning cylinder (6x8 mm)</td>
			<td style="width:173px;">Bellco</td>
			<td style="width:145px;">2090-00608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">L-15 Leibovitz culture medium</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">21083-027</td>
			<td>No phenol red</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:300px;">Polystyrene round bottom tube (5 ml) Facs tube</td>
			<td style="width:173px;">Falcon BD</td>
			<td style="width:145px;">352008</td>
			<td>No cap, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Falcon 2063 tubes (5 ml)</td>
			<td style="width:173px;">Falcon BD</td>
			<td align="right" style="width:145px;">352063</td>
			<td>Snap cap, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Nunc Lab-Tek 8 well coverglass</td>
			<td style="width:173px;">Thermo scientific</td>
			<td style="width:145px;">155409</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Charcoal-filtered FBS</td>
			<td style="width:173px;">Life technologies</td>
			<td style="width:145px;">12676011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">GW4064</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">G5172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">TCDCA</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">T6260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">CDCA</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">C9377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Other chemicals</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">n.v.t.</td>
			<td></td>
		</tr>
	</tbody>
</table>

real time monitoring of intracellular bile acid dynamics using a genetically encoded fret-based bile acid sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

The FRET-BAS sensor presented is based on the ligand binding domain of FXR (LBD-FXR) attached to two fluorophores citrine and cerulean) and an LXXLL motif. This sensor allows investigations into bile acid transport in living cells with high spatial and temporal resolution (Figure 1A). Mutations in cerulean and citrine were applied to promote the formation of the intramolecular complex (Figure 1B</...

Watch this Scientific Journal Video about Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor at JoVE.com

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

real-time-monitoring-intracellular-bile-acid-dynamics-using

Research

JoVE Journal

Bioengineering

9.8K Views.  Academic Medical Center. We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types. 

Video: Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...