Retaspimycin

Cell proliferation is studied using the cell proliferation reagent WST-1. Briefly, 8 × 10³ cells are seeded in triplicate in 96-well plates and treated for 5 days, with either trastuzumab or Retaspimycin as indicated. Viable cells are estimated on the basis of their ability to metabolize tetrazolium salt WST-1 to formazan by mitochondrial dehydrogenases. Quantification of the formazan dye directly correlates with the number of metabolically active cells and is analyzed by a scanning microplate reader. Results are shown as means ± SE^[3].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

RPMI-8226 cells are harvested from cultures grown in vitro in RPMI medium 1640 supplemented with heat-inactivated 10% (wt/vol) FBS and 100 units/mL penicillin/streptomycin at 37°C under a humidified 95%/5% (vol/vol) mixture of air and CO₂. Cells are washed twice by using sterile Hepes-buffered saline (HBS) and suspended in HBS to a concentration of 1 × 10⁸ viable cells per mL. Twelve female Nu/Nu nude mice (≈20 g) are used in the assay. RPMI-8226 cells (1 × 10⁷ cells per mouse) are implanted in the right flank. When tumor volume reaches ≈200-500 mm³ (≈4 weeks postimplantation), animals receive a single i.v. dose of 50 mg/kg Retaspimycin via the tail vein. At 4, 24, and 48 h posttreatment, the animals are killed with carbon dioxide, and tumors are removed and stored at −80°C until analyzed. Four animals are used for each time point. Tumor samples are homogenized in an ice-cold, nitrogen-sparged 1:1 solution of MeOH:150 mM citrate, 0.2% (wt/vol) EDTA, and 0.2% (wt/vol) ascorbate (pH 3.0) for 1 min in an ice/water bath with a homogenizer at 17,500 rpm. Samples are centrifuged for 5 min at 4°C at 18,000 × g. The supernatants are diluted 1:1 with ice-cold, nitrogen-sparged 75 mM citrate, 0.1% (wt/vol) EDTA, and 0.1% (wt/vol) ascorbate (pH 3) containing 25 ng/mL deuterated 17-AAG as internal standard and analyzed by LC-MS/MS analysis. The standard curve is prepared for Retaspimycin, 17-AAG, and 17-AG in 1:1 MeOH:150 mM citrate, 0.2% (wt/vol) EDTA, and 0.2% (wt/vol) ascorbate (pH 3.0); diluted 1:1 with ice-cold, nitrogen-sparged 75 mM citrate, 0.1% (wt/vol) EDTA, and 0.1% (wt/vol) ascorbate (pH 3.0) containing 25 ng/mL deuterated 17-AAG as internal standard; and analyzed by LC-MS/MS^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.