Fp 8300 irm spectrofluorometer

Nr8383 cells were cultured (5 × 10⁵/Petri dish) and exposed to CNTs (5, 10, 20 cm²/cm² for MWCNT/NM403 and 4.5, 9, 19, 38 cm²/cm² for SWCNT/NRCWE‐055) for 24 hours. Activity of caspase‐3 (CASP3) enzyme was determined using the EnzChek CASP3 fluorometric assay kit (molecular probes) with some modifications. In brief, cells were pelleted by centrifugation at 500 g for 5 minutes, washed twice with phosphate‐buffered saline and incubated in 50 μL lysis buffer on ice for 30 minutes. The lysate was centrifuged at 5000 g for 5 minutes, and then 50 μL of supernatant was added to 50 μL of the reaction mixture and incubated for 30 minutes at room temperature. Fluorescence of the reaction mixture was measured at excitation/emission wavelengths of 430/535 nm using the FP‐8300 Irm spectrofluorometer (Jasco).

After exposure, 10 μL of Alamar Blue^® Reagent (Roche) was added directly to each well. The plates were incubated at 37°C for 3 hours. Fluorescence was measured at excitation/emission wavelengths of 570/600 nm using a FP‐8300 Irm spectrofluorometer (Jasco, Lisses, France).