Preparation of cultured Sample Clauses

Preparation of cultured cells for toxicity assays For MTT assays, clear, flat-bottomed 96-well plates were used (Thermo Scientific, Loughborough, UK). For ATP assays, white, flat bottomed 96-well plates (Xxxxxxx Bio-One Ltd, Stonehouse, UK), pre-treated with 100 µL of 0.01% poly-L-Lysine (Sigma-Aldrich, Poole, UK), were used which were prepared as follows. Following sterilisation of the white plate with 70% ethanol, poly-L-Lysine was pipetted into each well and incubated for 1 hour, after which it was removed and the plate was allowed to dry. SH-SY5Y and S.NNMT.LP cells were trypsinised, pelleted and counted as described in Section 2.1.3. In order to minimise the time either cell line spent outside the incubator, all flasks were prepared one after the other rather than in parallel. This ensured that in studies comparing cell lines, both SH-SY5Y and S.NNMT.LP cells spent approximately equal time outside of the incubator prior to being seeded onto a plate. Following the cell count, cells were diluted to an appropriate density as described in each experimental chapter. One hundred µL of diluted cell suspension was then added to a number of xxxxx on a 96-well plate. In assays comparing SH-SY5Y and S.NNMT.LP cells, this process was then repeated for the remaining cell line, resulting in the plate layouts seen in figure 2.3. Each seeded plate was used to test 1 compound. 1 2 3 4 5 6 7 8 9 10 11 12 A SH SH SH SH LP LP LP LP SH* SH* B SH SH SH SH LP LP LP LP LP* LP* C SH SH SH SH LP LP LP LP D SH SH SH SH LP LP LP LP SH SH SH SH E SH SH SH SH LP LP LP LP LP LP LP LP F SH SH SH SH LP LP LP LP G SH SH SH SH LP LP LP LP H SH SH SH SH LP LP LP LP 1 2 3 4 5 6 7 8 9 10 11 12 A SH SH SH LP LP LP SH# SH# B SH SH SH LP LP LP LP# LP# C SH SH SH LP LP LP D SH SH SH LP LP LP SH SH SH E SH SH SH LP LP LP LP LP LP F SH SH SH LP LP LP G SH SH SH LP LP LP H SH SH SH LP LP LP
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