Pe conjugated anti mouse cd11b

Mortality and morbidity were checked once a day each working day. Animals were observed individually once a week for the recording of clinical signs and body weight. Blood samples for hematology analyses and DNA analyses were collected 16 weeks after transplantation and at termination. Erythrocyte count, mean cell volume, packed cell volume, hemoglobin, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocyte count, leucocyte count, differential white cell count with cell morphology, and reticulocyte count were determined using the Scil Vet ABC Plus⁺ analyzer (Scil Animal Care, Treviglio, Italy).
Cytofluorimetric analyses of PB were performed on a Becton Dickinson LSR II machine with BD FACSDiva software. PB from mice was analyzed for hematopoietic reconstitution using the following antibodies: phycoerythrin (PE)-conjugated anti-mouse CD11b, anti-mouse CD3, and anti-mouse B220 (BD Biosciences, San Jose, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD45.1 and allophycocyanin (APC)-conjugated anti-mouse CD45.2 antibodies (BD Biosciences, San Jose, CA) were used to evaluate donor-host chimerism at 16 weeks and termination.
Genomic DNA was extracted from the blood for determination of VCN at 16 weeks using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany).

Cells were washed twice in phosphate buffered saline (PBS) and re-suspended at 1 × 10⁶ cells/mL in FACS staining buffer (PBS/0.1% NaN₃/1% FBS). Cells were blocked with rat anti-mouse CD16/CD32 (BD Pharmingen, San Diego, CA, USA) at 4 °C for 5 min and then stained for 30 min with FITC-conjugated anti-mouse SRA, anti-mouse CD11b, anti-CD40, PE-conjugated anti-mouse CD11b, anti-mouse CD36, and anti-mouse CD86 (all BD Pharmingen) on ice in the dark. The cells were washed, re-suspended and analyzed on a Navios Flow Cytometer (Beckman Coulter, Brea, CA, USA). The data were processed using Kaluza software.