Coating Protocols for Stretch Chambers

Stretch chambers as sold are not sterile and provide no cell adhesion. Therefore, you must autoclave the chamber first for 20 minutes at 121°C, and then coat the membrane must with the extracellular matrix before seeding cells.

Coating protocol: Fibronectin

  1. Prepare a fibronectin solution by dissolving 0.05 mg/mL of fibronectin in PBS.
  2. Place the stretch chamber in a culture dish and pour the fibronectin solution into the chamber well so that it completely covers the bottom surface.
  3. Incubate the fibronectin-treated chamber in the culture dish at 37°C for at least four hours.
  4. Remove the culture dish from the incubator and draw up any remaining solution from the chamber using a pipette or other suitable device.

Coating Protocol: Collagen

  1. Prepare and autoclave a dilution of hydrochloric acid (pH3.0, 1 mM).
  2. Dilute type 1 collagen in the autoclaved hydrochloric acid.
  3. Place the stretch chamber in a culture dish and pour the collagen solution into the chamber well so that it completely covers the bottom surface.
  4. Cover the culture dish with a lid and incubate at 37°C for at least four hours.
  5. Remove the culture dish from the incubator and leave it to stand for a period. Then draw up the remaining solution from the chamber using a pipette or other suitable device.
  6. Rinse the chamber twice with serum-free culture fluid to remove any excess collagen solution that may have remained after the above steps.

Coating protocol: Gelatin

  1. Prepare a gelatin solution by dissolving 2% gelatin powder in PBS. Autoclave the gelatin solution.
  2. Place the stretch chamber in a culture dish and pour the gelatin solution into the chamber well so that it completely covers the bottom surface.
  3. Incubate the gelatin-treated chamber in the culture dish at 37°C for at least four hours.
  4. Remove the culture dish from the incubator and draw up any remaining solution from the chamber using a pipette or other suitable device.

The protocol of Fluorescent Staining after Stretching

  1. Splice out the cell plane from the stretch chamber with a surgical knife (as large as approx. 0.8 × 0.5 cm) – the size varies according to the size of the container to be used since step 2. Splicing to make the longer side get along with the stretching direction makes it easy to find out the stretching direction afterward.
  2. Put the membrane spliced in step 1 into the container with PBS(‐). [Using 1×1 chamber (without frame) attached to cover glass.]
  3. PBS(‐) wash, 2 times.
  4. Fix with 4% formalin solution in PBS(‐)->RT, 5 min, shake.
  5. PBS(‐) wash, RT, 5 min, 3 times.
  6. 0.1% TritonX-100 in PBS(‐), pierce cell membrane-> RT, 5 min, shake.
  7. PBS(-) wash-> RT, 5 min, 3 times.
  8. Block 2% BSA in PBS(‐)-> RT, 30 min, shake.
  9. The primary antibody in 0.1% Tween20 in PBS(‐)-> RT, 30min, shake.
  10. 0.1% Tween20 in PBS(‐) wash-> RT, 5 min, 3 times.
  11. The secondary antibody in 0.1% Tween20 in PBS(‐)-> RT, 30 min, shake.
  12. 0.1% Tween20 in PBS(‐) wash-> RT, 5 min, 3 times.
  13. Put on a sliding glass to enclose (Perma Fluor) – put the cell membrane side down.
  14. Fluorescent observation.

For more information, check out our blog article: Fluorescent Staining on a flexible PDMS membrane