In vitro toxicity testing
The LTA was performed as described previously.14,15Briefly, PBMCs were plated in flat-bottom 96-multi-well plates at a density of 1×105cells per well in quadruplicate and treated with a final concentration of 800 μM SMX or increasing concentrations of SMX-HA of 25, 50, 100, 200, 400, or 800 μM for 2 hr at 37°C in a 5% CO2humidified atmosphere. SMX and SMX-HA solutions were freshly prepared in dimethyl sulfoxide (DMSO) and diluted in culture media to give the desired final concentration of drug or RM (DMSO final concentration is always kept at ≤1%). A standard curve for measuring cell death was generated by seeding cells at 25%, 50%, 75% or 100% of cell concentration in culture media in quadruplicate. After incubation, drugs in solution were removed by centrifugation at 500 × g for 10 min. Then, cells were suspended in 100 μL fresh RPMI-1640 media supplemented with 10% FBS, 100U/mL penicillin G sodium and 100 μg/mL streptomycin sulfate, and left to recover for 18 hours in an atmosphere of 5% CO2 at 37°C. Cell viability was quantified using MTT staining as described previously.14
The iPTA was performed in similar fashion as the LTA (above) except that calcium free Locke’s solution was used as the medium. Platelets were isolated from blood samples as described above and plated at a density of 1× 107 cells/well. To pellet platelets after treatment plates were centrifuged at 900 × g for 15 min.