Each experiment was performed in triplicates and data are represented as mean SEM

Cl, pH 8.0, for 30 minutes. Zymosan was then washed twice with PBS and finally resuspended in PBS. After one more sonication step, FITC-labeled zymosan was divided in aliquots, frozen in liquid nitrogen, and stored at 220uC. Phagocytosis assays were performed in 12-well plates in which 100,000 cells were seeded per well the day before. Cells were incubated in control or FK866 containing medium for 3, 15, or 24 hours prior to assay and activated overnight with 100 ng/ml LPS. FITC-labeled zymosan particles were complement opsonized by incubation in FBS for 1 hour at 37uC, washed twice with PBS, and finally resuspended in serum-free control or FK866 medium. Cells were washed once with serum-free medium and incubated with 1 ml zymosan suspension for 30 minutes at 37uC. The particle-tocell ratio was approximately 10:1. Particle engulfment was terminated by washing cells twice with PBS and removing extracellular zymosan by treatment with 500 ml 100 U/ml lyticase for 10 minutes at room temperature. Successively, cells were Spreading Assay RAW 264.7 cells expressing Lifeact-EYFP were pre-incubated in control or 5 nM FK866 medium for 0, 3, 15, or 24 hours before they were harvested by treatment with 1 mM EDTA/PBS. After washing, the cells were suspended in medium with 1% BSA. A recovery period of 20 minutes at 37uC was allowed before cells were seeded in a fibronectin coated BD Falcon 96-well imaging plate at 4000 cells/well. Cell spreading was monitored for three hours by recording the increase in EYFP-pixel area per cell on a BD Pathway high-content spinning disc confocal microscope, using a 20x objective and 363 montage capture. In order to combine the data from three independent experiments, the time axes had to be synchronized. To achieve this, a Boltzmann simulation curve was produced for each data set HC030031 cost between time points 0 and 200 in steps of 2 minutes using OriginLab data analysis and graphing software. Data sets were then combined and analysed for statistical NAD+ Controls Macrophage Morphodynamics detached with 0.05% trypsin/0.5 mM EDTA, resuspended in 1 ml medium with serum, pelleted, and finally resuspended in 200 ml 1% paraformaldehyde in PBS. Samples were analyzed by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657107 FACS and phagocytosis efficiency was determined by measuring the fluorescence intensity of the FITC positive population as well as the percentage of cells in that population. The phagocytic index of each sample was calculated as the product of the mean FITC intensity of the positive population times the % of FITC positive cells. To determine the effect of nucleotide supplementation on the phagocytosis efficiency of control and FK866-treated cells, RAW 264.7 cells were incubated for 24 hours with 100 mM NAD+, 100 mM NMN or 50 mM NADP+ alone or in combination with 5 nM FK866 and phagocytosis efficiency was determined as described above. Adhesion and Internalization Assay Internalization efficiency was determined essentially as described by Sahlin et al.. Cells and zymosan particles were prepared as for the phagocytosis assay. After 30 minute incubation with serum opsonized zymosan at 37uC, plates were transferred to ice and wells were washed twice with ice cold PBS. Cells were then scraped in 1 ml cold medium, divided in two fractions, transferred to microcentrifuge tubes, and spun down. One fraction of each sample was resuspended in 0.05% trypan blue in 13 mM potassium dihydrogen citrate/saline, pH 4.4, to quench the fluorescence of all extracellular particles, and the