Cell Density

The optimal cell density must be determined. For transport and receptor function assays, a good confluent cell layer enhancing the target density is often advantageous, and cell numbers in the range of 8000 to 12 000 per 384 wells should be considered. The influence of cell number and variations on the signal must also be monitored. Functions such as sensitivity to drugs, transcriptional activity, permeability, cells in mitosis or the differentiation state often change with cell density. For these assays, semi-confluent cultures are often preferable.

Surface proteins (e.g., receptors) can be sensitive to proteases such as trypsin. Receptor function must be carefully checked if cells are to be used at different time-

points after seeding. It is recommended that cells are harvested using nonenzyma-tic solutions such as cell dissociation solution (Sigma) or HyQutase (Perbio).

For image analysis, and for localization assays, a good separation of the single cells is needed, and aggregates must be avoided. Larger cells (e.g., U2-OS cells) with a clearly separated, round nucleus and minimal morphological variation are ideal.

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