Note: Depending on the cell lines and the nature of the co-culture, the researcher can decide which side of the membrane is seeded first. To maintain sterile incubations of cells seeded on the underside of the membranes, use sterile Petri dishes for the Millicell® single-well inserts and sterile feeder trays for Millicell® 24- and 96-well cell culture insert plates.
1. Using an optimized seeding density, seed the first cell type in either the apical wells or on the basolateral underside of the membranes. Refer to the recommended working volumes chart (see page 42) for appropriate apical volumes. For basolateral seeding volumes, use approximately 200 ìL for Millicell® 12 mm single-well inserts and Millicell®-24 insert plates, and approximately 30 µL for Millicell®-96 insert plates.
2. Incubate in a 37°C CO2 incubator for 1 to 4 hours to allow the cells to attach to the membrane.
3. Gently turn the Millicell® device over and seed the second cell type in either the apical well or on the basolateral underside of the membrane.
4. Incubate in a 37°C CO2 in the incubator for 1 to 4 hours to allow the second cell type to attach.
5. Add appropriate volume of media to the apical or basolateral chambers and return to incubator.