F?rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. 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. .lsm or .lif files) directly in Image J using appropriate plugins (available at http://www.openmicroscopy.org) and move to step 4 4.1.14. For Stack 1: use Channel 01 (citrine). For Stack 2: use Channel 00 (cerulean). Click on Edit Selection Add to manager, to open the ROI manager window. Check the checkbox ‘Show All’. 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 Canagliflozin reversible enzyme inhibition 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. Select one cell. Click on Plugins 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 Canagliflozin reversible enzyme inhibition fluorescence intensity measured in both channels. If microscope setup-specific files (.lsm or .lif instead of .avi) files are used the channel order might be reversed. 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 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, Canagliflozin reversible enzyme inhibition do not use this graph when analyzing kinetic data, since the running average will also smooth fast kinetic responses. FRET measurements using Fluorescence Activated Cell Sorting (FACS) 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). 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. Count cells using the coulter counter or a counting chamber. Dilute pellet in FACS uptake buffer to a concentration of 1 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. Pipet 200 l cells per FACS tube and protect them from light. Add the desired concentration of the compound (bile acids, synthetic FXR ligands, transporter inhibitors). Vortex. Incubate for 20-30 min at RT while shaking (in the dark). Meanwhile, start the FACS (the lasers need time to warm up). Set the flow cytometry gating parameters for the experiment (see Figure 3): Load around 100,000-200,000 NucleoBAS or CytoBAS transfected cells to determine the gates. First adjust the FSC and SSC voltages to plot GFPT1 the cells in the center of the plot. 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. Set the correct gates (Gate P1 up to Gate P4), see Figure 4. 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. Use gate P2 in the FSC-H/FSC-A window to remove duplets.