Chemical Stability Tests

1. Determine what membranes and cells are to be used.
2. Remove desired membrane samples and take initial pictures for discoloration.
3. Soak membranes in methanol for 10 minutes.
4. Incubate culture media in culture plates for 15 minutes.
5. Remove membranes from methanol and gently wash in culture media.
6. Apply vacuum grease to membranes.
7. Pipette a small amount of media into each membrane trough to reduce the chance of bubbles forming when placed into media.
8. Insert membranes into media in plates, well side down.
9. Apply vacuum grease to cloning rings and place on membranes that require it, which are the membranes that will have cells on them.
10. Incubate during cell passaging.
11. Perform passaging according to protocol and desired dilution.
12. Pipette cells into cloning rings and wells.
13. Incubate.
14. Change media in all wells as required.

At a later time for imaging discoloration:

1. Remove culture plate from incubator.

2. Remove membranes for discoloration and wash with deionized water.

3. Wash with 70% ethanol and allow to dry.

4. Image and return to culture well.

At a later time for bead assay:

1. Remove culture plate from incubator.

2. Prepared 15 µm beads in deionized water (4 µL in 1.5 mL deionized water).

3. Pipette small volume of beads above membrane.

4. Return to incubator for a short time.

5. Remove and image both in focus with the membrane and below.