Measuring DT40 Cell Doubling Time and Immunoglobulin Gene Conversion

To determine DT40 cells doubling time, cultures of 5 × 10⁴ cells pr ml per condition were set and counted in duplicate by haemocytometer every 6 h for 4 days. Data were analysed with GraphPad Prism and doubling time was determined by nonlinear regression of data to Exponential (Malthusian) growth equation (Extended Data Fig. 8b). Immunoglobulin gene conversion was monitored by the conversion of IgM⁻ cells to IgM⁺ cells by flow cytometry. Fluctuation analysis was performed as described^{22 (link)} with modifications. In brief, 4 single-cell clones of DT40 WT or Fam72a^−/− were stained with anti-IgM-PE (1:40, clone M-1, SouthernBiotech) and 12 populations of 5 × 10⁴ IgM⁻ cells were sorted for each clone. The proportion of IgM-gain was measured by flow cytometry using the same staining after two weeks of expansion (same time of expansion) and five days later for the Fam72a^−/− cells (to achieve the same number of divisions as WT at two weeks). Data were analysed with FlowJo and GraphPad Prism.