Platelet quality control bound anti CD62P and anti CD63 antibodies were determined by analyzing 10,000 platelets for PE positive fluorescence. Biotin label based cytokine and chemokine microarray assay A custom mouse cytokine array kit was purchased. The array spotted the membrane with 50 specific antibodies against cytokines and chemokines per our request. For the assay, we followed the instructions precisely as stated by the manufacturer. Briefly, mem branes were placed in an eight well tissue culture tray and incubated with 2 mL of blocking buffer at room temperature for 30 min. After decanting the blocking buffer from each container, 1 mL of sputum supernatant from the sample was added and incubated overnight at 4 C.

After decanting the samples, all membranes Inhibitors,Modulators,Libraries were washed three times with 2 mL of wash buffer I at room temperature with shaking, followed by two washes with wash buffer II at room temperature with shaking. One milliliter of 1 250 diluted biotin conjugated antibodies was prepared and incubated for 2 h at room temperature, and the washing steps were repeated as before. Two milliliters of 1 1000 diluted Inhibitors,Modulators,Libraries horse radish peroxidase conjugated streptavidin was added, and the membranes Inhibitors,Modulators,Libraries were incubated for 2 h at room temperature, followed by additional washing. Spots were visualized using detection buffer C and D. Five hundred Inhibitors,Modulators,Libraries liters of a detection buffer C and 500 L of detection buffer D were mixed, loaded onto the membranes to cover the entire surface, and incubated for 5 min. The membranes were then covered in plastic wrap and exposed to radiographic film for 20 min the signal was detected using film developer.

Each film was scanned into an image processing and analysis program, and spots were Inhibitors,Modulators,Libraries digitized into pixel densities. The densities were exported into spreadsheet software and the background intensity was deducted before analysis. The data were normalized to the values for positive controls provided by the manufacturer as 100%. ELISA Blood samples were kept standing for 15 min until coagula tion. After high speed centrifugation, the supernatant was collected, aliquoted and stored at 70 C for analysis. The concentrations of thrombospondin, tissue inhibitor of metalloproteinase 1 and thymus chemokine 1 were measured using spe cific ELISA kits following the manufac turers protocols.

Standard curves and regression equations were calculated based on measurements of standard sam ples, and the cytokine concentrations in the serum samples were calculated according to the regression equations. so Statistical analysis Results are presented as the mean standard error of the mean. When the data were normally distributed, Bartletts test was used to test variance equality. Survival curve data were analyzed using the Kaplan Meier method and Wilcoxons test with SPSS 17. 0 for Windows.