Cd19 fitc

K562 CD34⁺ cells (1 × 10⁶ cells/mouse) were collected, and inoculated via tail vein injection into 6–9 week-old NOD/SCID mice, which was accepted sublethal irradiation (300 cGy). Two days later, the mice transplanted with CD34⁺ cells were randomized into 4 groups respectively(5 mice per group): (1)DMSO group as a negative control; (2) Baicalein administration alone(20 mg/kg); (3) IM administration alone (200 mg/kg); (4) Baicalein combined with IM. The mice were given an intraperitoneal(i.p.) injection with or without Baicalein (20 mg/kg) every other day. IM(200 mg/kg) was given orally once every day. The transplanted mice were euthanized after 6 weeks and bone marrow cells, blood cells and spleen cells were harvested. To evaluate leukocyte proportion of human cell engraftment, cells signed with antihuman CD45-PE antibody(eBioscience) were analyzed through flow cytometry. Human specific cell subpopulations were measured by marking with antibodies to human CD34-FITC (eBioscience), CD33-FITC (eBioscience), and CD19-FITC(eBioscience). Human CD45⁺ cells from BM of mice were harvested via immunomagnetic activated cell selection. To assess BCR-ABL expressing cells in malignant engraftment, selected CD45⁺ cells from BM were assessed for BCR-ABL mRNA expression by qRT-PCR.

Mice bearing tumors with a volume of 180–220 mm³ received 0.1 mg/kg DSR-6434 or saline (i.v.), and spleens were harvested 4 hr later. Splenocytes were isolated using a 90 µm cell strainer, red blood cells were lysed in Pharm Lyse™ (BD Biosciences, UK) and incubated with αCD16/CD32 Fc blocking antibody (eBioscience) followed by CD49 pan NK-FITC (5 µg/mL), CD69-PE (2 µg/mL), CD3ε-PECy5 (2 µg/mL; all from BD Pharmingen) and CD19-FITC (5 µg/mL; eBioscience). Samples were analyzed by flow cytometry (FACScanto, BD Biosciences) and analyzed as previously described.14 (link)

Retroorbital blood was collected and differential blood counts were obtained using an automated Bayer Advia 120 MultiSpecies Analyser (Bayer HealthCare). For flow cytometric analysis (FACS-Canto II; BD Bioscience), red blood cells were lysed with ammonium chloride buffer (0.150 mM NH₄Cl, 0.1 mM EDTA, 0.150 mM KHCO₃) for 10 min on ice. CLL cells were stained with CD19-FITC (1:100), CD5-APC (1:80), B220-eFlour605 (1:40), IgM-PECy7 (1:200) antibodies (eBioscience).