3 minutes at 4000 rpm and 4uC to pellet all cellular protein. Supernatants were neutralized with 140155 ml 2 M KOH/0.2 M KH2PO4, pH 7.5 and diluted 1:10 in water. Per well, 100 ml diluted PCA extract was added to 100 ml CellTiter-Glo reagent and the ATP concentration was determined using an ATP standard series. For determination of total cellular protein, pellets were dissolved in 250 ml 1 M NaOH and heated for 30 minutes at 95uC. Protein concentration was then measured in 1:50 diluted NaOH extracts. NADH Autofluorescence Measurements Cellular autofluorescence was determined as a measure of mitochondrial NADH-level. Cells were seeded in glass-bottom WillCo MedChemExpress 946128-88-7 dishes and incubated with or without 5 nM FK866 for 24 hours. Using the 340 nm filter block, cells were exited for 500 ms and autofluorescence was recorded of at least 10 different fields using the Olympus IX-71 wide field fluorescence microscope equipped with an EM-CCD camera. Per field analyzed, the background fluorescence as well as the fluorescence of each cell in the field was measured by determining the average grey value of two regions of interest in the background of the image and of two regions of interest in the cell body. Per cell, the autofluorescence was determined by subtracting the average background fluorescence from the average fluorescence inside the cell. Per condition, 80110 cells were analyzed. Oxygen Consumption Measurements Mitochondrial respiration was assessed by measuring oxygen consumption on an Oroboros Oxygraph-2 k respirometer according to a standard protocol provided by the manufacturer. Control and 24 hour FK866 treated cells were analyzed in parallel in two separate chambers of the respirometer. After air calibration of control or FK866 medium in the chambers and stabilization of the signal, 16106 cells were injected into the respective chambers. Basal respiration rate was measured at the point where the O2-flux signal stabilized. Oligomycin was added to each chamber and the leak respiration rate was determined after stabilization of the signal. Next, 7 mM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone was added to reach maximal oxygen consumption in the cells. Finally, 30 nM rotenone was added and after stabilization of the system, the residual oxygen consumption could NAD+ Controls Macrophage Morphodynamics be determined. The data was analyzed using the DatLab software provided with the instrument. Proliferation Assay For cell proliferation measurement, the protocol developed by Skehan et al. was used. Cells were seeded in four 96-well plates in 100 ml culture medium and incubated overnight. At T0, the medium of plates T6, T15, and T24 were replaced with medium containing 0, 1, 5, or 10 nM FK866 and plate T0 was fixed for sulforhodamine B staining of protein content. After 0, 6, 15, and 24 hours of FK866 treatment, cells were washed twice with cold PBS and fixed in 10% trichloroacetic acid for 1 hour at 4uC. After fixation, plates were washed five PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19655565 times with water and stored at 220uC until all plates were collected. Cellular protein was stained with 50 ml 0.5% SRB in 1% acetic acid for 20 minutes after which wells were washed four times with 1% acetic acid. Plates were dried at 60uC for 3 hours, protein was dissolved in 150 ml 10 mM Tris-HCl, and the absorbance of each well was measured at 510 nm on a BioRad Benchmark Plus micro plate reader. Values were corrected for background SRB staining by subtracting the average absorbance value of wells th