Gr1 pe

Spleens of immunized mice were harvested at indicated time points and single cell suspensions were obtained by passing through 70 μm cell strainer (Greiner Bio-ONE, cat. 542070). Single cells were suspended in FACS buffer (2% FBS in PBS) and incubated with antibody at 4°C. To distinguish the Qβ-specific B cells in germinal center, cells were firstly stained with Qβ-AlexaFluoro 488 and PNAbio (Vector Laboratories, cat. B-1075), and then Fc-receptors were blocked with anti-mouse CD16/CD32 (BD Bioscience, cat. 553142). Finally, streptavidin-APC Cy7 (BD Bioscience, cat. 554063) and anti-mouse CD38-PerCP Cy5.5 (Biolegend, cat. 102722) were applied to determine germinal center cells, anti-mouse B220-PE Cy7(BD Bioscience, 552772) for B cells, anti-mouse IgM-PE (Jackson ImmunoResearch, cat. 115-116-075), IgD-PE (eBioscience, cat. 12-5993-83), CD4-PE (BD Bioscience, cat. 553653), CD8-PE (BD Bioscience, cat. 553032), CD11b-PE (BD Biosience, cat. 553311), CD11c-PE (BD Biosience, cat. 553802), GR1-PE (BD Biosience, cat. 553128) to exclude other cell types (CD4, CD8: T cells; CD11b: monocytes and macrophages; CD11c: dendritic cells; GR1: neutrophils) and immature B (IgM⁺IgD⁺) cells.

All animal experiments were performed under an approved protocol (IACUC 2008–0155) from the Augusta University Institutional Animal Care and Use Committee. 6–8-week-old female BALB/c mice were used for all engraftment experiments. Donor BM cells from BALB/c mice were isolated and transduced with various constructs prepared in the MIG retroviral vector as described previously [18 (link)–20 (link)], then 5 × 10⁶ cells were transplanted into lethally irradiated (800 cGy), syngeneic hosts via tail vein injection. Tumor cell engraftment and progression was monitored weekly beginning two weeks after i.v. injection by examination of the levels of the GFP+ population in peripheral blood using standard flow cytometry analysis. Antibodies used were, B220-APC (#553092), IgM-PE (#553409, CD4-APC (#553051), CD8-PE/Cy7 (552877), Gr1-PE (553128), Mac1-PerCP/Cy5.5 (#550993), Sca1-PE/Cy7 (558162), Kit-APC (553356) and Flt3-APC (#560718) from BD Biosciences (Franklin Lakes, NJ). For secondary and subsequent sequential transplantations, 1–2.0 × 10⁶ BM cells from primary and subsequent diseased mice, respectively, were again transferred by tail vein injection into sub-lethally irradiated (600 cGy) female BALB/c recipient mice.

To measure the dynamic changes in the proportions of immune cell populations in the peripheral blood of rats, 50 μl of blood samples was taken for flow cytometry analysis. After lysing red blood cells, the remaining leukocytes were resuspended in PBS containing 2% FBS, and then were stained with HIS48-FITC (eBioscience), CD11bc-allophycocyanin (BioLegend), CD68-PE (Biolegend) or CD206-PerCP (Abcam) for 30 min at 4°C. On the other hand, mice cells were analyzed by staining with CD11b-FITC (BD Bioscience), Gr-1-PE, Ly6G-APC (BD Bioscience), Ly6C-PerCP-Cy5.5 (eBioscience), F4/80-PE (eBioscience) or CD206-PerCP (Biolegend, #141716). Acquisition of flow cytometry data from the BD FACSCantoII flow cytometer was performed using FACSDiva software (BD Biosciences). The number of events analyzed was 10,000 per sample. Analysis was performed using FlowJo software (Tree Star).

Mouse lungs were minced to single cell suspensions and filtered through 70‐μm cell strainers (BD Biosciences, San Jose, CA) as previously described.9, 28 The erythrocytes were lysed using red blood cell lysis buffer (BD Biosciences) and washed with phosphate‐buffered saline. Cells were labeled with monoclonal antibodies including anti‐mouse CD45‐FITC, Gr‐1‐PE, CD11b‐PerCP, CD48‐APC, and counting beads (BD Biosciences) was added. Matched isotype antibodies were used as negative controls. All samples were analyzed using a FACSCalibur cytometer (BD Biosciences) and CellQuest software, and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

At indicated time points, mice were intraperitoneally anesthetized, tracheotomized, ventilated and intracardially heparinized as described [24] (link). Blood was taken from the caudal Vena cava and lungs were perfused with 0.9% NaCl via the pulmonary artery. Bronchoalveolar lavage was performed twice with 800 µl ice-cold PBS. After spinning, supernatant was snap frozen for cytokine analyses. Total leukocytes were counted manually on Neubauer Chamber and differentiated by fluorescent-activated cell sorter (FACS) analysis (FACS Calibur, BD Biosciences, Heidelberg Germany) using forward versus side scatter characteristics and the specific antibodies CD45 PerCP (clone 30-F11, BD Biosciences), GR-1 PE (clone RB6-8C5, BD Biosciences) and F4-80 APC (clone BM8, Invitrogen, Karlsruhe, Germany) as described [25] (link). Total blood leukocytes were counted and differentiated by FACS analysis using BD TruCOUNT Tubes, forward versus side scatter characteristics and specific antibody staining with CD45 PerCP and GR-1 PE [25] (link). Cytospins from BALF were obtained by centrifugation of 100 µl BALF cell suspension at 20×g for 10 minutes (Cytospin 3, Shandon Ltd, Runcorn, UK) and subsequently stained with May-Grünwald Giemsa.