LC/Electrospray Ionization/MS/MS Analysis. To measure plasma concentration of different CY metabolites, 40 m l of each sample was injected onto a C-18 column (2 × 150 mm, 5 m m ODS, 100A; Phenomenex, Rancho Palos Verdes, CA) at a flow rate of 0.2 ml/min. The separation was performed using a gradient starting from 50% methanol over 4 min, then to 100% methanol over 2 min, followed by 100% methanol for 7 min. The HPLC column effluent was introduced onto a Quattro Ultima triple quadropole MS (Micromass). Analyses were performed using electrospray ionization in positive-ion mode with multiple reaction monitoring. The transitions monitored were mass-to-charge ratio (m/z): m/z 261® 140 for CY, 259® 140 for 4-OH, 221® 140 for PM, 199® 171 for DCE, 293® 221 for CB, and 261® 154 for ifosfamide (internal standard).

Real-Time PCR Analysis. Cyp3a13 mRNA quantitation was performed by using the following probe and primers: Cyp3a13-probe: FAM-CTT CAC AAA CCG GCG GCG CTT-TAMRA; Cyp3a13-For: ATG GCT GGT GAA GGA ATG TTA CTC T; and Cyp3a13- Rev: TTT CAA AAT ACC CAC TGG ACC AA. Relative mRNA abundance was calculated using the comparative delta-Ct method with GAPDH mRNA as standard.

Hepatocyte Preparation and Coculture Experiments. Mice were perfused through hepatic vein with 10-15 ml of 0.5 mM EGTA in PBS, followed by 25 ml of 0.05% collagenase in serum-free DMEM. The liver was removed and filtered through 70-m m nylon mesh; cell suspension was loaded on the Percoll step gradient (20-40%) and centrifuged at 1000 × g for 15 min. The yield of hepatocytes (sediment at the bottom of the gradient) was, on average, 4-5 × 10⁷ cells per liver with viability > 90%.

Flow Cytometry. Bone marrow cells were flushed from both femurs with a small amount of PBS (~2 ml of bone) by using a 21 G syringe, resuspended by pipetting, and pelleted. Spleen cell suspension was obtained by filtering the tissue through the nylon (70 m m) mesh in a small amount of PBS and pelleted. Red blood cells were lysed by resuspending the pellet in 1 ml of buffer: 0.15 M NH₄Cl/0.1 mM KHCO₃/0.1 mM Na₂EDTA for 1 min at room temperature. Cells were washed once with PBS.

After removal of red blood cells, bone marrow cells and splenocytes were resuspended in the staining buffer (Dulbecco’s PBS with 0.1% BSA); 10 × 10⁵-cell aliquots were incubated on ice in 100 m l of 1:1000 dilution of rat serum (Rockland, Gilbertsville, PA) for 30 min, washed with PBS-BSA, and stained with FITC-labeled anti-mouse B220 and PE-labeled CD3 antibodies (Pharmingen) according to manufacturer’s protocol. After a 1-h incubation, cells were washed three times, resuspended in staining buffer, and analyzed by flow cytometry using a FACSCalibur and FACSComp Software (BD Biosciences, San Jose, CA). Sample data were collected on 10,000 ungated cells. In each experiment, bone marrow cells from untreated mice were used to determine the fluorescence window specific for the B and T cells, and these windows were used to gate the cell population when analyzing the experimental samples to determine the percentage of B220- (or CD3)-expressing cells.