Uis259l

For cell culture studies, MWCNT stock (2 mg/ml) were prepared in Ca²⁺/Mg²⁺-free phosphate-buffered saline (PBS), pH 7.4, according to the methods previously described (Pacurari et al., 2008 ; 2016 ). Briefly, MWCNT were prepared in PBS followed by indirect sonication at 4°C for 5 min (Hielscher ultrasonic processor, UIS259L) at amplitude 100% and cycle 1. After indirect sonication, the suspension was directly sonicated at 4°C for 1 min at 5W output and 10% duty cycle (Branson Sonifier 450). The stock solution (2 mg/ml) of MWCNT was kept at 4°C and used within 2–3 weeks. Prior to cell culture experiments, the MWCNT stock solution was directly sonicated for 1 min at the setting indicated above. Samples that were used for cellular studies were freshly prepared from the stock on the day of cell treatments to desired concentrations in RPMI containing 0.1% FBS and resonicated by a direct ultrasonication for 1 min before use.

For cell culture exposures, MWCNT were prepared in DM. Transmission electron micrographs of MWCNT dispersed in DM determined that DM promoted significant dispersion of MWCNT. MWCNT were prepared in DM, followed by indirect sonication at 4°C for 5 min (Hielscher ultrasonic processor, UIS259L) at amplitude 100% and cycle 1. Following indirect sonication, the suspension was directly sonicated for 5 min at 5W output and 10% duty cycle (Branson Sonifier 450). The stock solution (0.5 mg/mL) of MWCNT was kept at 4°C and used within 2–3 weeks. Prior to cell culture experiments, the MWCNT stock solution was directly sonicated for 1 min at 5W output and 10% duty cycle.

For cell culture studies, ND-MWCNT were prepared in dispersion media (DM) consisting of PBS (pH 7.4, Ca/Mg-free) supplemented with 0.6 mg/ml mouse serum albumin and 1.1 mg/ml 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as previously described (Porter et al., 2008 ). DPPC was prepared fresh as a 1 mg/ml stock solution in 100% ethanol. Transmission electron microscopy (TEM) micrographs of MWCNT dispersed in DM demonstrated that DM promotes significant dispersion of MWCNT (Porter et al., 2008 ). ND-MWCNT and MWCNT-7 were prepared in DM by indirect sonication at 4 °C for 5 min (Hielscher ultrasonic processor, UIS259L; Ringwood, NJ) at amplitude 100% and cycle 1. Following indirect sonication, the suspension was directly sonicated for 5 min at 5 W output and 30% duty cycle in 1 min increments (Branson Sonifier 450; Danbury, CT). The stock solution (0.5 mg/ml) of ND-MWCNT or MWCNT-7 was kept at 4 °C and used within 2–3 weeks. The ND-MWCNT and MWCNT-7 stock solutions were directly sonicated for 1 min at 5 W output and 30% duty cycle prior to cell culture experiments. Cells were treated with DM, ND-MWCNT, or MWCNT-7 at 1.2 mg/ml, a concentration which was extrapolated from previous murine in vivo exposure experiments based upon alveolar surface area (Porter et al., 2010 (link)).