2 ��M Wortmannin, 25 ��M PD98059, 10 ��M U0126. Stock Enzastaurin purchase solutions of each reagent SKI 606 selleck compound were Inhibitors,Modulators,Libraries individually prepared in DMSO and the final concentration of DMSO in the culture medium was 0. 1% in all treatments, includ ing controls. At least two independent experiments were performed, with five biological replicates in each experiment, for all results described here. Samples were stored at 80 C until analysis. ELISA Microarray Analysis Concentrations of individual proteins in CM and cell lysates were quantitatively measured using sandwich enzyme linked immunosorbent assay, as pre viously described in detail. Briefly, ELISA microarray chips were custom manufactured using aminosilanated, 25×75 mm glass slides stamped with a hydrophobic barrier that was used to cre ate 16 wells on each slide.
Inhibitors,Modulators,Libraries The ELISA reagents used in these analyses have been previously evaluated and shown to have no cross reactivity Inhibitors,Modulators,Libraries and to be able to quantita Inhibitors,Modulators,Libraries tively detect purified antigens that were spiked into human Inhibitors,Modulators,Libraries serum. The capture antibodies were sus pended in PBS at concentrations ranging from 0. 5 to 1. 0 mg/ml. These antibodies were printed using a GeSiM noncontact NanoPlotter NP2 printer. Sixteen identical chips were printed on each slide, such that each chip was isolated by a hydrophobic barrier. Each capture antibody and control reagent was printed in quadruplicate on each chip. Successful printing was confirmed using the RedReflect capability on the ScanArray ExpressHT laser scanner.
Inhibitors,Modulators,Libraries The printed slides were blocked with 1% casein in PBS at 4 C and, Inhibitors,Modulators,Libraries after washing, stored desiccated and under vacuum at 20 C until use.
Inhibitors,Modulators,Libraries In order to generate standard curves Inhibitors,Modulators,Libraries for each of the ELISA analyses, a single mixture containing all the anti gens was prepared in 0. 1% casein in PBS and containing 100 pg/ml green fluorescent protein. This stock solution of the standard mixture was aliquoted and stored at 80 C, and an aliquot was thawed on a daily Inhibitors,Modulators,Libraries basis for Inhibitors,Modulators,Libraries each ELISA microarray analyses. For this analysis, the stan dard stock solution was serially diluted 3 fold to create at least 7 dilutions of the standard mixture. Each dilu tion, and an antigen free blank, was analyzed on three seperate chips.
Several Inhibitors,Modulators,Libraries incubation steps are included in processing the ELISA microarray chips.
Washes were performed between each incubation step by submerging Inhibitors,Modulators,Libraries the Inhibitors,Modulators,Libraries slides in PBS containing 0. 05% Tween 20.
Either 20 ul of the standard mixture or an individual, diluted sam ple was then pipetted onto each of three replicate chips. Samples were then incubated overnight in a closed chamber with saturated humidity and gentle mixing. Slides were then incubated Inhibitors,Modulators,Libraries with a cocktail of all Calcitriol mechanism detec tion antibodies sellckchem selleck Imatinib Mesylate in 0. 1% casein/PBS buffer for 2 h. The level of biotinylation was then increased using the bioti nyltyramide amplification system. Finally, slides were submerged in 1 ug/ml of Cy3 or Alexa 647 conjugated streptavidin in PBS T, and incubated for 1 h in dark.