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How to seed cultured cells for single clone selection?

Single clone selection is necessary when performing stable cell line production or drug resistant cells.  The goal is to dilute cells down to a concentration such that when they are plated in a dish there is a single cell per well.  The single cell clone is expanded by continually increasing the well size until freezer stocks can be established.

Theoretically, by knowing the starting cell number of the suspension a scientist could simply make a single dilution down to 1 cell per well and proceed.  However, due to pipetting variances and slight changes in cell viability, this method rarely produces successful clones.  The best route for clonal selection is to plate a slightly higher cell number in a well and then perform 1:2 serial dilutions across a 96-well plate.  Eventually, one of the wells will have a single cell.  Keep in mind that single clone selection is difficult, so replicates of the dilution series are recommended.  Growth of multiple single clones allows researchers to have biological replicates of resistant cell lines in downstream studies.

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