See Microfluidic Permeability Assay for In Situ Monitoring of Endothelial Barrier Properties for background information.

**Materials**

- Appropriate cell line grown to confluency on µSiM device (see appropriate µSiM Device Cell Culturing Protocol)
- Appropriate Cell Culturing Media
- Fluorescein Isothiocyanate (FITC) Dextran, 10 kDa, or desired fluorescent small molecule
- Pipets (P200, P20) and tips
- Amber 1.5 mL Eppendorf tubes (or cover with foil)
- Nikon Ti2 Epifluorescence microscope (or equivalent)
- CytoVU (SiMPore)
- Beakers for tip waste and media waste

**Methods**

- Prepare media and FITC Dextran dilutions.
- Warm media in 37ºC water bath.
- Make 2 mg/mL and 1 mg/mL solutions of 10 kDa FITC Dextran (200 and 100 µM).
- Protect from light.
- Note: Concentration for assay is 1 mg/mL. The 2 mg/mL solution will be diluted when adding to the device. The 1 mg/mL solution is to be used to obtain the initial concentration image for normalization.
- Note: Concentrations may need to be optimized on different scopes for proper dynamic ranges and to avoid photobleaching during experimentation.

- Set up device.
- Remove old media from device and wash one to two times with media, top and bottom channels.
- Remove media from top channel and add 50 µL fresh media.
- Be sure there are no bubbles in the device.

- Place device on microscope in CytoVU.
- Set to 40X (long working distance) objective.
- Set channel to phase and focus on device membrane or cell monolayer. Center the membrane in the image. Take image.
- Prepare microscope for image acquisition.
- Select for time-lapse with 11 images in 1 minute intervals.

- Move window to left side and focus on window edge, so the line between the window and support is crisp.
- For trench down devices, then focus 70.6 µm below the cell monolayer. This will focus on 50 µm from the window edge along the trench, where we measure fluorescent intensity in the analysis.

- Add 50 µl of 2 mg/ml dye solution to the top well and take time-lapse images.
- After first image is acquired, make sure everything looks normal.
- Let it run for 10 min. You will see dye entering the bottom channel over time

- Add 1 mg/mL dye solution to bottom channel and take source concentration image. The dye should uniformly flood the channel.
- If the device moves, refocus and realign window.

#### Analysis

- Relabel images to end in 1-12 (1 is for time zero, 11 is after 10 min, 12 is source concentration). The source concentration image must have the same name as the others to run in MATLAB. Save as .tif files.
- Determine pixel number for 50 µm from trench.
- Open in ImageJ and measure distance from edge of image to edge of window. Be sure measurement is pixel number or convert to pixels.
- Round to nearest whole number for MATLAB.

- Open TIF2PLOT in MATLAB.
- Open images to Current Folder using ‘Browse to Folder’ in MATLAB.
- Run TIF2PLOT (‘Add to Path’ if necessary).
- Enter information as prompted
- Ex for name: Device1_f_
- Ex for pixel: 280

- Save plot of I
_{x}/I_{0}vs time - Record y values given in MATLAB (these are the actual values for I
_{x}/I_{0}at each time point)

- Enter information as prompted

- Open in ImageJ and measure distance from edge of image to edge of window. Be sure measurement is pixel number or convert to pixels.
- Determine permeability coefficient.
- Open ‘E-12Sweep’ Excel file.
- Enter y values from MATLAB into green cells under ‘Experimental Data’.
- Divide values by 100 so they are no longer percentages.

- Find highlighted cell (lowest root mean square error (RMSE), aka best fit from COMSOL simulation) and write down corresponding D value.
- If highlighted cell is first or last, go to corresponding excel file for that range.
- If first cell, P is lower, use ‘E-13Sweep’.
- If last cell, P is higher, use ‘E-11Sweep’.

- Calculate permeability.
- P = D/L, P = monolayer permeability (m/s), D = adjusted diffusion coefficient (m
^{2}/s), L = length (m).- D from excel is given in m
^{2}/s. - Length in our system is 10 µm, or 10 x 10
^{-6}m.

- D from excel is given in m
- Report permeability in cm/s.
- Multiply by 100 for final value.

- P = D/L, P = monolayer permeability (m/s), D = adjusted diffusion coefficient (m

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