Organotypic raft culture of primary human keratinocytes (PHKs) Notes Use 106 keratinocytes per raft. Typical Protocol Trypsinize the PHKs off the flask and resuspend in (say) 2ml raft medium. See separate protocol for raft medium Remove the medium from the collagen plugs and overlay with the new medium containing PHKs. Incubate for 1-2 days to allow the PHKs to settle and become confluent. Autoclave the following; Spatulas for scooping out the collagen plug PBS Stainless steel mesh grid in a glass petri-dish. (Thoroughly rinse the glass petri-dish to remove detergent. Make the four 'legs' for the mesh grid by placing it flat onto a microfuge tube rack and pressing a tube into an edge, deforming it downwards. This downward deformed bit is a leg.) When the PHKs are confluent:- Separate the edge of the collagen plug from the wall of the well by going round it with a sterile scalpel. Allow the collagen plug to contract away from the walls for a few hours. This is like cutting round a cake in a cake tin. The PHK 'membrane' may contract from the edge also and detach a little from the collagen Remove media, scoop out plug and deposit onto the mesh in the petri-dish, keeping everything sterile. Optional - rinse in PBS before placing onto mesh This transfer is the trickiest part of the whole procedure. There is no easy way to do it although I found that the collagen gel is stronger than it looks. If you get it intact, right side up (ie. PHKs up), and looking roundish on the mesh, without having scraped off all the PHKs, then you've done well. Usually it ends up on the floor. Sometimes the PHKs all come away like a membrane; try to spread it back onto the collagen. Keep sterile Feed the raft by adding 12-15ml of raft medium to the petri-dish. The idea is to wet the underside of the mesh but not to let the liquid come onto the upper surface. If the top of the raft becomes wet it will destroy the gradient and spoil the experiment. Watch out for air bubbles loitering under the raft. Refeed every two days People seem to leave them for 8-14 days to grow/differentiate Add in BrdU 8-12 hours before harvesting (see separate BrdU protocol) Suppliers Stainless steel wire mesh 3 1/16 inch 40 mesh .010 (=wire size) 304 SS circle, item #105493 100 for about $25 from; US Filter - Johnson/Niagara Screen (a civil engineering, water filter type company) Niagara Mesh Sales Fax 001 713 939 1337 Tel 001 713 939 1830

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