ALine Modular Device: in situ Small Molecule Permeability (Confocal Microscope)

See 10 kDa Small Molecule Permeability Optimization 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)
  • Andor Dragonfly Confocal microscope (or equivalent)
  • CytoVU (SiMPore)
  • Beakers for tip waste and media waste

Methods

  1. Prepare media and FITC Dextran dilutions.
    1. Warm media in 37ºC water bath.
    2. Make 2 mg/mL and 1 mg/mL solutions of 10 kDa FITC Dextran (200 and 100 µM).
      1. Protect from light.
      2. 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.
      3. Note: Concentrations may need to be optimized on different scopes for proper dynamic ranges and to avoid photobleaching during experimentation.
  2. Set up device.
    1. Remove old media from device and wash one to two times with media, top and bottom channels.
    2. Remove media from top channel and add 50 µL fresh media.
    3. Be sure there are no bubbles in the device.
  3. Place device on microscope in CytoVU.
  4. Set to 10X objective.
  5. Set channel to phase and focus on device membrane or cell monolayer. Center the membrane in the image. Take image.
  6. Prepare microscope for image acquisition.
    1. Select for time-lapse with 11 images in 1 minute intervals.
  7. Focus on the window edge, so the line between the window and support is crisp. Take an image for reference to the window position.
    1. For trench down devices, then focus 100 µm below the cell monolayer.
  8. Add 50 µl of 2 mg/ml dye solution to the top well and take time-lapse images.
    1. After first image is acquired, make sure everything looks normal.
    2. Let it run for 10 min. You will see dye entering the bottom channel over time
  9. Add 1 mg/mL dye solution to bottom channel and take source concentration image. The dye should uniformly flood the channel. For trench down devices, you are focused within the trench, and will only see dye in that region, not in the support region.
    1. If the device moves, refocus and realign window.

Analysis

  1. Open images in ImageJ. Use in focus image to find x position for the center of the window.
  2. On timelapse file, draw line down center of window and measure fluorescence intensity for each image.
  3. On source concentration image, draw line down center of window and measure fluorescence intensity.
  4. Normalize timelapse images to source concentration image to get percent fluorescence intensity at each timepoint. Subtract fluorescence intensity of  time zero image as background.