Apc mouse anti human cd14

The phenotype of MSC was confirmed prior to cell delivery by flow cytometry. Cells suspended in staining buffer containing 1% BSA and 0.05% sodium azide in D-PBS were incubated with Rat BD Fc Block (BD Biosciences, USA) for 5 min., and later with fluorochrome-conjugated antibodies: APC anti-human CD29, FITC anti-human CD44, BV421 anti-human CD90, APC-anti-human CD105, BV421 anti-human CD73 (BD Biosciences, USA), BV570 anti-human CD45 (Biolegend, USA), APC mouse anti-human CD34, APC mouse anti-human CD14 (BD Biosciences) for 40 min. Following washing with a cell staining buffer, cells were fixed with 1% neutral buffered formalin overnight. Samples resuspended in 1% BSA were assessed on the following day using a BD LSR II cell analyzer (Becton Dickinson, USA).

Microarray analysis was carried out at the University of Maryland Greenebaum Comprehensive Cancer Center Flow Cytometry Shared Service (FCSS). Induced macrophages were fixed in paraformaldehyde, washed, and incubated in blocking buffer consisting of PBS, human Fc Block (0.5 mg/mL, BD), 8% FBS, and 0.01% sodium azide. Cells were then incubated with APC Mouse Anti-Human CD14, APC Mouse IgG2α, к Isotype Control, PE Mouse Anti-Human CD68, or PE Mouse IgG2b к Isotype control (BD, San Jose, CA, USA) in buffer containing PBS, 0.2% saponin, 8% FBS, and sodium azide, washed, and kept at 4 °C until fluorescence-activated cell sorting analysis. Data were acquired by flow cytometry using a BD FACS Canto II Cell Flow Cytometry System and analyzed using FCS Express software (De Novo, Pasadena, CA, USA).

Thymidine, PMA and propidium iodide were purchased from Sigma-Aldrich (St Louis, MO, USA); FAM48A (p38IP), p38, p21, GCN5, green fluorescent protein (GFP), γ-Tubulin, β-actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Phospho-p38 mitogen-activated protein kinase (Thr180/Tyr182) was purchased from Cell Signaling Technology (Danvers, MA, USA); PE Mouse anti-human CD11b/Mac-1 and APC Mouse anti-human CD14 were purchased from BD Pharmingen (San Diego, CA, USA); CD14+ microbeads (human) was purchased from Miltenyi Biotec, Bergisch Gladbach, Germany; Recombinant Human M-CSF (cyt-308) and murine M-CSF was purchased from Prospec protein specialists (East Brunswick, NJ, USA). As GFP antibody is also reactive against yellow fluorescent protein, it was used for detection of yellow fluorescent protein.

Infection of human blood ex vivo was performed as previously described [17 (link)] using FITC-labeled bacteria. Briefly, indicated concentrations of FITC-labeled bacteria in 100 μL of DPBS were combined with 1 mL of freshly drawn human blood containing heparin (Fresenius Kabi; 20 USP units/mL) in 2.0-mL microcentrifuge tubes and incubated at 37°C with end-over-end rotation. Preliminary experiments in which sodium citrate was used as an anticoagulant yielded results similar to those with heparinized blood. At indicated times, red blood cells were lysed using erythrocyte lysis buffer EL (Qiagen) following the manufacturer’s protocol (QIAamp RNA Blood Mini-Handbook), and leukocytes were resuspended in 200 μL ice cold DPBS. Pelleted cells were divided into 50-μL aliquots, stained with APC Mouse Anti-Human CD3, APC Mouse Anti-Human CD14, APC Mouse Anti-Human CD19, or PE Mouse Anti-Human CD66c (BD Biosciences), and analyzed using flow cytometry (FACSCalibur, BD Biosciences). Forward and side-scatter were used to set gates on leukocyte populations (lymphocytes, monocytes, and granulocytes). B cells were defined as CD19⁺ leukocytes, T cells as CD3⁺ leukocytes, monocytes as CD14⁺ leukocytes, and PMNs as leukocytes expressing high levels of surface CD66 (CD66^High).