Time Saving Procedures On Anti-cancer Compound Library

, 2011). Mouse serum EPO and Lcn2 levels were Anti-cancer Compound Library measured by ELISA (DuoSet ELISA Kit, R&D Systems) according to the manufacturer's instructions. Anti-PyNL-specific antibody responses were measured as previously published ( Coban et?al., 2010). Blood, spleens, and bones were collected from mice on the appropriate days, and single-cell suspensions were prepared. Cells were treated with ACK-lysis buffer (Sigma-Aldrich) before staining. All antibodies were from BD Biosciences otherwise mentioned. Cells were FcR-blocked with anti-CD16/CD32 monoclonal antibody prior to staining. Cell surfaces were stained with phycoerythrin (PE)-conjugated anti-TER119, fluorescein isothiocyanate-conjugated (FITC) anti-CD71 (C2), and PE-Cy7 Gr1 (RB6-8C5, Biolegend) antibodies. Samples were acquired on a BD LSRFortessa Flow Cytometer and analyzed with FlowJo software. Liver and spleen tissues were homogenized, total RNA was isolated with RNAeasy Mini Kits (QIAGEN), and ReverTra Ace (Toyobo) was used for reverse transcription according to the manufacturer's instructions. The cDNA fragments were amplified by Real-Time PCR Master Mix (Toyobo), and fluorescence was detected by a 7500 Real-Time PCR System (Applied Biosystems). The relative induction of each mRNA expression level of each gene was normalized to the expression level of 18S RNA. The primers for 18S rRNA, Lcn2, Hmox1, IFN��, and IL-1�� were purchased from Assays on Demand (Applied Biosystems). Livers and spleens were removed and fixed with 4% paraformaldehyde (PFA) for 4?hr at 4��C. Samples were then equilibrated in sucrose, embedded in OCT compound click here (Sakura Finetek, Japan), and kept in liquid nitrogen. Sections were cut with a cryostat (Leica) and mounted onto slides (Matsunami MAS-GP, Japan). After removing the OCT compound in PBS, sections were permeabilized and blocked overnight. Primary antibodies were as follows: anti-mouse Lcn2/NGAL (R&D Systems), PE-F4/80 (BM8, BD Biosciences), FITC-Gr1 (RB6-8C5, BD Biosciences), and HO-1 (Stressgen SPA 895). Ceramidase Secondary antibodies were as follows: Alexa Fluor 594-donkey anti-rat IgG antibody, Alexa Fluor 488-donkey anti-rabbit IgG antibody, and Alexa Fluor 594-donkey anti-rat IgG antibody (Invitrogen). The sections were coverslipped with mounting medium and observed with a fluorescence microscope (Zeiss, Germany). Tissue iron staining was performed by using iron staining kit (Accustain Iron Stain, Sigma). Differences between two groups were analyzed for statistical significance using a two-tailed, unpaired Student's t test if the data passed normal distribution analysis (Sigma Stat). If not, the statistical significance of differences between two groups was analyzed by a nonparametric Mann-Whitney test. Differences between three groups were analyzed by a Kruskal-Wallis test with Dunn's multiple comparisons. For survival curves, the log-rank (Mantel-Cox) test was performed. p?