t r o u b l e s h o o t i n g One corner of the membrane is cut to provide a reference point for the position of the drops loaded on the membrane surface. Removing membrane from Petri dish during sterilization and/or drying is unnecessary and increases chances of contamination. Note that one of the membrane surfaces is shiny, the other matte. It is necessary that the matte membrane surface contact the support during drying; the shiny surface must face up. If the shiny surface comes in contact with the support during drying, the membrane will stick to the support and it will require re-wetting with sterile water to lift it. It is essential that the membrane surface on which the drops are to be placed remain unwetted prior to plating. If that surface is accidentally wetted, the membrane can be restored to usefulness by rinsing it with sterile water and then drying it completely. Membrane must float as a raft in supporting media before plating. When plating cells on the membrane surface do not place the drops too closely to one another to prevent accidental bridging of the drops, or too closely to the edge of the membrane to prevent spill over. When co-culturing two different cell lines, one cell line in the drops and another cell line in the Petri dish itself, the following procedure has to be followed: 1) add supporting media to Petri dish to float membrane; 2) add first cell line to supporting media keeping pipette away from membrane to prevent cross contamination; 3) add second cell line in drops to membrane surface. To change media always change supporting media first and media in drops after. For co-culturing of cell lines the use of (SFM) Serum Free Media is preferable. For an arrangement where the membrane rests on a wetted filter paper, do not wet filter paper to the point that the level of liquid floats the membrane off the filter paper. Remove supernatant from drops before removing membrane from filter paper. When changing supporting media keep pipette away from membrane at all times. Change media in Petri dish before changing media in drops on the membrane surface. When changing media in drops, change media one site at a time and make sure pipette tip does not come in contact with the membrane. Replace exact amount of media to original drop site. When expanding original drop site area it is preferable that the final volume is equal to no more than double the original volume in which the cells were plated. The recommended final volume should not exceed 500 ul. When transferring cells while these are still attached to the membrane surface, make sure to put membrane face down in the new Petri dish and add only enough media to cover membrane. Membrane should not float in media, this will prevent transfer and attachment of cells to the new Petri dish. It is critical to remove media from drops before transfer. When cutting out a piece of the membrane (i.e. containing a selected sub-population) follow instructions for transfer as described above. To trypsinize remove media in drops before removing supporting media. Wash membrane very gently with PBS before adding trypsin. Add media to trypsin to stop reaction and collect cells.