Gibco™ RPMI 1640 Medium

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	supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 50 U/mL penicillin and 50 µg/mL streptomycin (complete medium, CM)

Protocol tips
supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 50 U/mL penicillin and 50 µg/mL streptomycin (complete medium, CM)

Publication protocol

Cells resuspended in RPMI-1640 (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 50 U/mL penicillin and 50 µg/mL streptomycin (complete medium, CM), seeded at a density of 2×104 cell/mL in 96-well plates and incubated at 37°C in 5% CO2 were exposed for 48 hours to incremental concentrations of α-bisabolol (dissolved in ethanol 1∶8; Sigma-Aldrich, St. Louis, MO) to determine the half maximal inhibitory concentration (IC50) for each cell population. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT (Sigma-Aldrich) incorporation as previously described [20], [21] and was expressed as ratio of number of cells treated with α-bisabolol to number of cells treated with vehicle alone. To compare the differential sensitivity to α-bisabolol of blasts vs normal cells, flow cytometry analysis was carried out in three selected BCR-ABL+ ALL patients, whose bone marrow samples contained 10 to 20% of residual normal T-lymphocytes. Samples were treated with 20, 40, 80 µM α-bisabolol for 24 hours, then immunostained with anti-CD10 APC, anti-CD3 FITC and anti-CD19 PE (Becton Dickinson, San Jose, CA) monoclonal antibodies (moAbs). At least 5×104 cells of each sample were acquired on a FACSCanto cytometer (Becton Dickinson) and subjected to PolyChromatic Plot analysis by FlowJo 9.3.3 software (Tree Star, Ashland, OR).



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