Anti human cd11b pe

RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY). The CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (Madison, WI). Anti-human CD11b-PE and Apoptosis Detection Kit I were purchased from BD Bioscience (San Diego, CA). Antibodies against ERK, phospho-SFK Y416, c-Raf, cleaved caspase-3, and anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Beverly, MA). Antibodies for western blot against LYN and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

All of the reagents, including VPA, were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. The CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (Madison, WI), and RPMI 1640 medium and fetal bovine serum (FBS) from GibcoBRL (Grand Island, NY). Annexin V-FITC Apoptosis Detection Kit I, PI/RNase staining buffer, anti-human CD11b-PE, anti-human CD14-PE and mouse IgG₁-PE were purchased from BD Biosciences (San Diego, CA). DRAQ5 was purchased from Abcam (Cambridge, MA). The Apoptosis Antibody Sampler Kit, anti-p27^kip1, CDK4, CDK6 and cyclin D1 were purchased from Cell Signaling Technology (Beverly, MA). All of the inhibitors, including the mitogen-activated protein kinase (MAPK) inhibitors (U0126, PD98059, SB203580 and SP600125), caspase-3 inhibitor (Z-DEVD-FMK) and caspase-9 inhibitor (LEHD-CHO), were obtained from Merck Millipore (Billerica, MA). The ApoTarget Caspase-3 Protease Assay Kit for caspase-3 activity and CasGLOW Fluorescein Active Caspase-9 Staining Kit were purchased from Invitrogen (Camarillo, CA) and eBioscience (Atlanta, GA), respectively, and the Immun-star WesternC Kit was purchased from Bio-Rad (Hercules, CA). Finally, the Western antibodies, anti-p21^cip1, CDK2, cyclin E, β-actin and anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Staining of digested single cell suspensions of lung tissue and blood leukocytes was performed for 15 minutes using fluorescently labelled antibodies: PerCP rat anti-mouse CD45 (30-F11 clone, 1:100, 557,235, BD), FITC anti-mouse CD11b (M1/70 clone, 1:100, 101,206, Biolegend), PE-Cy™7 rat anti-mouse Ly6G (clone 1A8, 1:100, 560,601, BD), APC anti-mouse Ly6C (clone HK1.4, 1:100, 560,595, BD), FITC rat anti-mouse CD3 (clone 17A2, 1:100, 561,798, BD), PE-Cy™7 rat anti-mouse CD4 (clones RM4–5 clone, 1:100, 552,775, BD), APC-H7 rat anti-mouse CD8a (clone 53–6.7, 1:100, 560,182, BD), APC rat anti-mouse CD19 (clone 1D3, 1:1000, 550,992, BD), PE rat anti-mouse CD49b (clone DX5, 1:1000, 553,858, BD), PE anti-mouse F4/ 80 (BM8 clone, 1:1000,123,110, Biolegend) and Alexa Fluor 647-labelled anti-mouse Msr1 (2F8 clone, 1:1000, MCA1322A647, AbD Serotec, NC, USA). Staining was performed with PE anti-human CD11b (ICRF44 clone, 1:1000, 555,388, BD), PE-Cy7 anti-human CD66b (G10F5 clone, 1:1000, 305,116, Biolegend) and APC anti-human Msr1 (7C9C20 clone, 1:1000, 371,905, Biolegend) stained human blood. All assays were performed by FACS Canto II cytometer (BD). Cell sorting was performed using FACSAria. Data were analysed using FlowJo software (Tree Star, Inc., Ashland, OR).

The hWJ-MSCs that were used were obtained from passage 3 at 80% confluence. The cells were detached from cell culture flasks (Falcon^®, Coring, Inc., New York, NY, USA) using trypsin/ethylenediaminetetraacetic acid (EDTA) solution 0.05% (Gibco^TM Thermo Fisher Inc., Waltham, MA, USA) and washed with PBS. Then, the hWJ-MSCs were incubated for 30 min at room temperature with saturating concentrations of the following monoclonal antibodies labeled with fluorochromes: PerCP/Cy5.5 anti-human CD 105, FITC anti-human CD90, APC anti-human CD 73, PE anti-human CD 34, PE anti-human CD 45, PE anti-human CD19, PE anti-human CD 11b, PE/Cy7 anti-human HLA-ABC, and PE anti-human HLA-DR (all purchased from BD Biosciences (San Jose, CA, USA). After removing the excess antibody through 3 washes with PBS, the hWJ-MSCs were ana-lyzed according to standard protocols using a flow cytometer (FACS Calibur™, BD Biosciences, San Jose, CA, USA) and FlowJo software (Becton, Dickinson & Company, Franklin, NJ, USA). The cell population was identified, and whether cells were positive or negative for each marker was determined based on the criteria of the International Society for Cell and Genetic Therapy (ISCT).