Cell seeding

Process map

Cells at well edgeCells containing the transgene are typically seeded using one of the following three approaches:

  • limiting dilution cloning
    Suspension cells are plated into liquid selection media at approximately 0.3 cells per well, or are sequentially diluted such that wells contain no cells, one cell or more than one cell. 
  • fluorescence-activated cell sorting (FACS) 
    Suspension cells are plated at one cell per well post-sort; efficiency varies from 30-70%.
  • clone picking from semi-solid media
    Cells are grown into colonies in semi-solid media with selection. They are then picked manually or using robotic equipment and transferred to the wells of microtitre plates. Afterwards cells must re-adapt to liquid culture.

With any of these methods, including the single-cell cloning methods, there remains a degree of uncertainty as to whether the resulting colony is monoclonal (derived from a single cell). However, by employing the Cell Metric CLD™ at this early stage for both limiting dilutions and FACS-based approaches, rapid screening of the entire population can be achieved immediately after seeding while still at the single cell level. In this way the Cell Metric CLD™ fully documents the evidence for single-cell deposition and hence monoclonality (see Clone screening).

The next step

Seeded microplates are taken forwards for clone screening to identify monoclonal populations.

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