Spheroid Optimisation Protocol
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Step 1 Preparing cells for seeding
If an enzymic dissociation step (such as trypsinisation) is required ensure that cells are washed twice in fresh media as per your normal protocol. Spin cells down into a pellet and carefully remove all/or as much supernatant as possible. Re-suspend cells in pre-warmed 1x happy cell media at a density of 100,000 cells per ml.
Step 2 Cell Seeding
Titrate cells at densities ranging from 10,000 to 1000 cells /100ul/well into low binding clear bottom 96-well plate and incubate for a minimum of 72 hours at 37°C/ 5% CO2 and 95% humidity. Check plates using microscope with low power objective at least once daily to assess optimal seeding density and incubation times.