The vertical lines correspond to the 10th and the 90th percentiles, and the circles, to the outliers

The vertical lines correspond to the 10th and the 90th percentiles, and the circles, to the outliers. MFC for the routine characterization of DLBCL cells and tumors samples for research and diagnostic/prognostic purposes. species was regularly monitored. 2.2. Flow Cytometry Analysis Expression of 27 normal and pathological B lymphoid markers (CD19, CD20, FMC7, CD22, CD23, Kappa, Lambda, CD10, CD5, CD38, CD27, CD39, CD43, CD62L, CD81, CD200, BCL2, BCL6, Ki67, IgM, LAIR1, CD123, CD11c, CD25, CD103, CD71, and CD180) Morusin was evaluated by labeling the 16 DLBCL cell lines with specific antibodies. Surface staining of the cell suspension was performed at the recommended volume per test in the dark at room temperature (see supplementary Table S1 for references and combinations). Among the markers that were studied, BCL2, BCL6, and Ki67 were evaluated by intra-cytoplasmic staining, using the fix and perm solutions of the kit GAS-002 (Nordic-Mubio?, Susteren, The Netherlands). Flow cytometry data were acquired with a Canto II flow cytometer (Becton Dickinson?, Le-Pont-de-Claix, France). The instrument setup and calibration were in accordance with the EuroFlow standard operating procedures [11,12]. For each marker, the mean intensity of fluorescence was evaluated after the initial gating of the cells of interest based on the CD45/SSC (side scatter) plot to exclude cell debris. Singlets were included in the FSC-H/FSC-A (forward scatter) plot, and events were analyzed, according to the EuroFlow panel guidelines. With the standard antibody panel, DLBCL-derived cell lines with a B-cell phenotype were selected based on the CD19/CD3 plot (or CD20/CD3 plot, if CD19 was weakly expressed). The total B-cell population represented the OR Boolean gate between the kappa-positive or lambda-positive B cell population. Events present on the kappa/lambda diagonal were removed [13]. For the other lymphoid markers, the total B-cell population gated on CD19 (or Morusin CD20) represented the AND Boolean gate. 2.3. Cell Viability Assay DLBCL cells were cultured in 96-well flat-bottom microtiter plates in the appropriate medium with FBS in the presence of increasing concentrations of mafosfamide (Santa-Cruz Biotechnology, Dallas, TX, USA), doxorubicin or etoposide (Selleckchem, Houston, TX, USA), or the BCL6 inhibitor 79-6 (Calbiochem, San Diego, CA, USA) for 4 days. The number of viable cells was determined using the Cell Titer Glo Luminescent Cell Viability Assay from Promega (Promega Corp, Madison, WI, USA). This test is based on the quantification of cellular ATP, as a marker of metabolically active Morusin cells, using a Centro LB 960 luminometer (Berthold Technologies, Bad Wildbad, Germany). Data were expressed as the mean percentage of six replicates and were normalized Morusin to the untreated control. 2.4. Gene Expression Profiling and Statistical Analyses Gene expression microarray data were from four independent cohorts of patients with DLBCL. The first cohort (i.e., training cohort) included 233 patients treated with R-CHOP (R-CHOP Lenz cohort). Results were validated using the CHOP Lenz cohort (= 181 patients treated with CHOP) [14], the Melnick cohort (= 69 patients) [15], and the FFPE R-CHOP cohort (= 72 patients) [7]. The patients pre-treatment clinical characteristics were previously described in References [7,14,15]. Affymetrix gene expression data are publicly available via the online Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession numbers “type”:”entrez-geo”,”attrs”:”text”:”GSE10846″,”term_id”:”10846″GSE10846 (Lenz Rabbit Polyclonal to STEA3 cohorts), “type”:”entrez-geo”,”attrs”:”text”:”GSE23501″,”term_id”:”23501″GSE23501 (Melnick cohort) and “type”:”entrez-geo”,”attrs”:”text”:”GSE53786″,”term_id”:”53786″GSE53786 (FFPE R-CHOP cohort) (Affymetrix HG-U133 plus 2.0 microarrays). Data were normalized with a Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings, and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500. Expression profiling data (accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE56315″,”term_id”:”56315″GSE56315) of DLBCL samples (= 73) and normal centrocyte and centro-blast samples (= 5/each) from human tonsils [16] were also compared. One or several probe sets were available for 24 of the 27 B-cell markers evaluated by MFC. In the presence of several probe sets for the same gene, the probe set with the highest coefficient of variation was retained (Supplementary Table S1). Overall survival (OS) differences between groups were calculated using the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model and Genomicscape (http://genomicscape.com) [17]. Survival curves were plotted using the Kaplan-Meier method. All analyses were done with R.2.10.1 and.