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. 2019 Nov 13;2(4):85. doi: 10.3390/mps2040085

Figure 3.

Figure 3

Updated experimental design for the HIMA. Primary HUFs are isolated and grown at 3% oxygen. Prior to the HIMA treatment conditions must be optimised. For this purpose, cells are treated with the mutagen of interest and cytotoxicity, DNA damage response, genotoxicity (i.e., DNA adduct formation) and/or lacZ mutagenicity are used to determine treatment conditions for the HIMA. For the HIMA primary HUFs are seeded on six-well plates (P1) and treated with the test agent using the optimised treatment conditions. Treatment is conducted at 3% oxygen. Cells are then serially passaged under standard culture conditions (20% oxygen) until they undergo senescence (P5+). Cells will eventually emerge from senescence and can be developed into immortalised clonal cell lines (P8+). The Nutlin-3a counter-screen is used to screen immortal clones for the presence of TP53 mutations. Only TP53-mutated clones (i.e., Nutlin-3a resistant clones) will be expanded and subsequently subjected to TP53 sequence analysis.