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.