A Histopaque 1077/1119 (Sigma Aldrich, St. Louis, MO) discontinuous gradient was created by placing 10 ml of Histopaque 1119 into a 50 ml tube, overlaying Ceritinib price 10 ml of Histopaque 1077, then overlaying 4 ml of blood/PBS mixture. The gradient mixture was centrifuged at 700g for 30 min at room temperature. Cell layers were removed from the plasma/Histopaque 1077 interface

(mononuclear cells) and from the 1077/1119 interface (heterophils) with a Pasteur pipette, combined and transferred to a 15 ml tube. Cells were washed by adding 6 ml of PBS and centrifuged at 600g for 10 min at room temperature. Supernatant was discarded and the cell pellet washed a second time with PBS. Supernatant was discarded and PBL resuspended in 1.5 ml of RNAlater Epigenetics Compound Library price (AM7021) (Applied Biosystems, Foster City, CA). Cells were refrigerated in RNAlater for 7 day then excess RNAlater was decanted and cells were stored at −80 °C until RNA isolation. RNA samples were isolated using the Ambion MagMax-96 kit for Microarrays (AM1839) (Applied Biosystems, Foster City, CA). Briefly, PBL were added to 0.6 ml of TRI Reagent Solution (Ambion, Austin, TX). Samples were homogenized and split into two 300 μl aliquots, with 1 aliquot further processed and the other held

in reserve. Samples were then processed using the Spin Procedure according to manufacturer’s instructions. Total RNA was Org 27569 eluted with 30 μl of Elution Buffer and stored at −80 °C. Quality and quantity of RNA were assessed by Nanodrop (Thermo Scientific, West Palm Beach, FL). The microarray data for this experiment have been deposited in NCBI’s Gene Expression Omnibus (GEO) [5] and [19] database and are accessible through the series accession GSE31387〈http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31387〉. Gene expression was assessed utilizing the global 2-color chicken 44K Agilent Microarray [43]. A total of 40 samples were hybridized to the microarray, using one individual from each of the

ten treatment groups from each of 4 independent experimental replications. Samples were arranged in a reference design, using the NV–NC-day 1 sample as the reference for each experimental replicate, on 36 arrays. Within each replicate, the NV–NC-day 1 sample was hybridized to the other 9 treatment groups. Dye assignments were swapped between replicates. Briefly, 400 ng of total RNA was reverse transcribed into cDNA with a T7 promoter region incorporated, then transcribed back into cRNA labeled with either Cy3 or Cy5 dye. Before hybridization, 825 ng of each labeled sample, Cy3 and Cy5, a blocking agent and fragmentation buffer were mixed together and incubated for 30 minutes at 60 °C. Following incubation, gex hybridization buffer was added and samples were hybridized to the microarray slide for 17 h at 65 °C.