CometChip Electrophoresis Starter Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 2×10^5 cells/ml		DNA damage can be analyzed using the IMSTAR Pathfinder system

Upstream tips
Seed 2×10^5 cells/ml
Downstream tips
DNA damage can be analyzed using the IMSTAR Pathfinder system

Publication protocol

DNA strand breaks levels were quantified by the alkaline comet assay as previously described [24] and reported according to the Minimum Information for Reporting on the Comet Assay (MIRCA) recommendations. After cell exposure to nanomaterials, for 6 or 24 h, cell media were discarded, and cells (A549 or THP-1a) were washed twice with PBS. Then, 200 μl of trypsin were added to each well, the plates were incubated for 4 min, after which 300 μl of freezing medium (80 % FBS and 20 % DMSO) was added to each cell sample. Cells were diluted to 2 × 105 cells/mL and stored at -80 °C. Embedding of cells in agarose was performed as previously described. Briefly, frozen cell samples were thawed quickly at 37 °C and suspended in agarose at 37 °C with final agarose concentration of 0.7 %. Cells were embedded on 20-well Trevigen CometSlides at 4 °C, after which these were placed in electrophoresis buffer for 40-min alkaline treatment. Electrophoresis was run with 70 mL/min circulation (5 %) of the solution for 25 min with applied voltage at 38 V (1.15 V/cm in the whole electrophoresis tank) and current of 294 mA. The slides were neutralized in Tris buffer (2 × 5 min), fixed in ethanol for 5 min and dried on a warm plate at 45 °C for 15 min. Cells on slides were stained in 40 mL/slide bath with TE buffered SYBR Green fluorescent stain for 30 min, dried at 37 °C for 10 min after which UV-filter and cover slips were placed on slides. DNA damage was analyzed using the IMSTAR Pathfinder system. The results are presented as averaged % tail of DNA of all cells scored on each Trevigen CometSlide. All slides included A549 cells exposed to PBS or 45 μM H2O2 as negative and positive controls for the electrophoresis.

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