Cells were washed twice to remove unbound antibody and resuspended in 100 μL FACS-PBS. Binding of primary antibody was detected using optimum concentration (determined by titration) of appropriate isotype-specific fluorochrome-labeled secondary antibody or avidin:anti-mouse IgM-phycoerythrin (PE) and anti-rat IgM-PE Adriamycin and anti-mouse IgG3–fluorescein isothiocyanate (Jackson Laboratory) and streptavidin (Caltag Medsystems). After incubation for 40 minutes at 4°C, cells were washed twice and finally resuspended in 250 μL FACS-PBS.
Unstained cells and cells labeled with secondary antibody alone were included as controls. Dead and apoptotic cells and debris were excluded from analysis using an electronic “live” gate on forward scatter and side scatter parameters. Data for up to 25,000 “live” events were acquired for each sample using a FACSCalibur cytometer equipped with 488 nm and 633 nm lasers and analyzed using
CellQuest software (Becton Dickinson). RNA was isolated using RNeasy kit (Qiagen) following manufacturer’s instruction and DNA was removed by the treatment with deoxyribonuclease (Qiagen). Protein Tyrosine Kinase inhibitor Complementary DNA was synthesized using 2 μg total RNA with reverse transcriptase (Roche Diagnostics) in a 20-25 μL volume. Polymerase chain reaction (PCR) was carried out as described.1, 5 Primer sequences and PCR conditions are provided in Supporting Table 1. The quantitative PCR analysis was carried out as described in Hay et al.5 For teratoma formation, cells were harvested, washed once with DMEM/F12, and mixed with Matrigel (BD Biosciences) and collagen.8, 9 Cells (1 × 106) were injected intramuscularly into immune-compromised NSG (NOD [nonobese diabetic] SCID [severe combined immunodeficient] gamma) mice. Teratomas formed within 6–8 weeks, and paraffin sections were stained with Masson’s
trichromatin 上海皓元医药股份有限公司 for all histological determinations. Cells were fixed with chilled methanol (−20°C) for 10 minutes, washed with PBS, and blocked with 10% goat serum and 0.02%–0.1% Triton X-100 for 1 hour. The cells were then incubated with primary antibody at the appropriate dilution at 4°C overnight. Secondary antibody was applied for 30 minutes after washing with PBS. The cells were finally mounted with Mowiol (Calbiochem) and then visualized and captured using a Leica DM IRB microscope. Enzyme-linked immunosorbent assays (ELISAs) were carried out as previously described.5 CYP1A2 and CYP3A4 activity was assessed using the pGlo kit (catalog numbers V8771, V8901; Promega) according to manufacturer’s instruction for nonlytic CYP450 activity estimation. CYP activities are expressed as relative light units (RLU/mL) per of media, normalized against percentage of hepatocyte-like cells.