Most HTS are plate-based assays involving one washing or separation step, as well as several pipetting steps. Good adherence of the cell line used is necessary, as the uncontrolled loss of cells is a frequent reason for low assay reproducibility. The degree of negative charges on the cell at physiological pH seems to be one important factor for good adherence. In addition, the presence of bivalent cations (Mg2+, Ca2+) and the presence of extracellular proteins determine the adherence of cells [26]. Examples of good adherent cell lines in standard culture plasticware are Chinese hamster ovary (CHO), baby hamster kidney (BHK), COS and NIH-3T3 cells. Poor adherence can be improved by coating the wells with charged substrates such as poly-L- or -D-lysine, or with components of the cellular matrix. Examples include fibronectin or collagen for fibroblasts, and laminin for epithelial cells; alternatively, a special plastic such as CellBind (Corning) can be used. In our experience, coating is necessary for cells such as HEK293.

0 0

Post a comment