Bd553062

After treatment, all mice were weighed, blood were collected by cardiocentesis under general anesthesia using a disposable syringe with needle (20-gauge), and liver and spleens were dissected and weighed (23 (link)). Collected blood (1 ml/mouse) was lysed with 1X Pharm Lyse™ lysing buffer (BD Biosciences) to destroy red blood cells and leukocytes, as described previously (21 (link)). Following centrifugation at 1,500 × g for 15 min at 4°C, white blood cells were collected and stained with phycoerythrin (PE)-labeled anti-mouse CD3 (BD553062; BD Biosciences, San Jose, CA, USA), PE-labeled anti-mouse CD19 (BD553786; BD Biosciences), fluorescein isothiocyanate (FITC)-labeled anti-mouse CD11b (BD553310; BD Biosciences) and FITC-labeled anti-mouse Mac-3 (BD553324; BD Biosciences) antibodies at 1:12 dilution of each for 30 min at 4°C. All samples were analyzed by flow cytometry (BD Biosciences) and quantified using CellQuest software version 5.2.1 (BD Biosciences), as previously described (21 (link)).

The blood and spleen of each mouse was collected. Splenocytes were isolated from the spleen in order to evaluate NK cell activity, as previously described (25 (link),26 (link)). To analyze blood samples (0.2 ml each) for cell markers, red blood cells were lysed using 1X Pharm Lyse^™ lysing buffer (BD Pharmingen, San Diego, CA, USA), according to the manufacturer's instructions. After centrifugation at 1,500 × g for 15 min at 4°C, white blood cells were collected and stained with phycoerythrin (PE)-labeled anti-mouse CD3 (BD553062, BD Biosciences, San Jose, CA, USA), PE-labeled anti-mouse CD19 (BD553786, BD Biosciences), fluroscein isothiocyanate (FITC)-labeled anti-mouse CD11b (BD553310, BD Biosciences) and FITC-labeled anti-mouse Mac-3 (BD553324, BD Biosciences) antibodies (the dilution of each antibody was 1:12) for 30 min at 4°C. The cells were washed with PBS and analyzed for cell marker population by flow cytometry, as previously described (25 (link),26 (link)).