Synchronous Morphogenisis on Filters

Procedure

1. Count Cells. Wanted: 1.5x10^8 cells.
2. Spin the cells in Steenšs clinical centrifuge, 1500 rpm for 4 min.
3. Aspirate media from the cell pellet. Resuspend the cells in 10 ml of LPS and spin as before.
4. Wash again in 10 ml LPS, then resuspend in LPS so that the total final volume is 750ul.
5. Place two filter paper circles cut to size in one 15mm (small) plate.
6. Add 3.2ml of LPS to filters. Allow liquid to soak in and remove any excess by aspiration.
7. Rinse a filter (Millipore, HA, 0.45um) in small beaker of LPS. Place on top of filter papers, dark side up. (Note: filters can be found in the fridge or cold room. They have been briefly boiled in LPS and stored under liquid)
8. Add 500ul of the LPS/cell mixture to the filter in a swirling pattern (moving outward) to create an even lawn of cells..
9. Place in sealed container (or saran wrapped) with a bit of water or wet paper towels (to keep humidity high).
10. Observe periodically for following 24 hours w/ microscope or magnifier.

LPS

• KCl, 3g
• MgCl2.6H20, 1g (=1ml)
• NaH2PO4, 7.5g
• Na2HPO4, 6.75g
• water to 2L
• Adjust pH to 6.4 and store at 0-5oC