H dimensional data generated by multicolour flow cytometry require an unbiased

H dimensional data generated by multicolour flow cytometry require an unbiased and rapid analysis, difficult to S the disease progresses it transitions into being hormone independent and perform using the conventional manual gating strategy; a ten colour panel generates 1024 theoretical cell populations (210) to be analysed in a bidimensional space. We have therefore applied a computational analysis pipeline approach to our dataset. flowType [21] was used to extract 6560 cell populations from every FCS file. flowMeans [19] was used as the population identification algorithm. The IL-10+, IL-17A+, and Foxp3+ populations were too small to be automatically identified. For these markers, all of the samples were combined into a single file to allow a more robust population identification using a static gate. The measured immunophenotypes were analyzed using receiver operator characteristic (ROC) curves. A cumulative distribution function (CDF) of the area under the curve (AUC) values is illustrated in Figure S4A. The immunophenotypes with an AUC score of higher than 0.9 were selected for analysis using RchyOptimyx [23] (Fig. 3A). To include all of the single-marker immunophenotypes, the CD4+, Foxp3+, IL-17A+, and CD8+ cellLyoplate Flow Cytometry for Biomarker DiscoveryFigure 2. Conventional and lyoplate based flow cytometry platforms have comparable intra- and inter-assay variability. A. Comparison of intra-assay variability between conventional- and lyoplate based- flow cytometry platform (CFP and LFP respectively). Coefficient of variation (CV) was calculated for each sample from experimental triplicates at one time point. Arrows indicate the origin of daughter cell populations. Each dot corresponds to one individual, horizontal bars represent medians. B. Comparison 1315463 of inter-assay variability between CFP and LFP. Cell frequencies obtained from the same leucocyte cone sample run across four different experiments. Percentages of IFN-c+, IL-10+, and IL-17A+ cells were calculated within memory CD4+ T cells (identified as live CD3+CD4+CD45RO+ cells). T regs were identified as live CD3+CD4+CD25highFoxp3+ cells. Average and standard error (ER) are indicated in the two bottom rows. doi:10.1371/journal.pone.0065485.gmedium-throughput processing of the samples, using pre-filled 96 well plates and a plate loader. Moreover, pre-formatted lyoplates, containing the same batch of reagents, can be reliably used through the entire duration of a study and across multiple centres. Therefore, LFP reduces hands-on time, while promoting automation and reagent standardization that are of primary importance in translational and clinical research studies. Our data indicate that lyophilized reagents resulted in more powerful cell stimulation and better marker discrimination, possibly due to improved reagent stability after lyophilisation. In keeping with the increased detection of IFN-c+, IL-10+, Foxp3+ and CD25+ cells, most of the lyophilized antibodies also resulted in increased resolution sensitivity as determined by a higher stain index (SI) on stained PBMC. Of note, tandem dyes PE-Cy5 and APC-H7 showed a decreased SI on stained PBMC compared to cells stained with liquid counterparts, indicating that lyophilisation might have a different impact on different fluorochromes. This aspect should be considered when Title Loaded From File designing the antibody cocktail to be lyophilized, and a pre-test of the lyophilisation impact onto the specific antibody-fluorochrome combinations should be performed, especially for tandem dye conjugates. If possible,choosing an.H dimensional data generated by multicolour flow cytometry require an unbiased and rapid analysis, difficult to perform using the conventional manual gating strategy; a ten colour panel generates 1024 theoretical cell populations (210) to be analysed in a bidimensional space. We have therefore applied a computational analysis pipeline approach to our dataset. flowType [21] was used to extract 6560 cell populations from every FCS file. flowMeans [19] was used as the population identification algorithm. The IL-10+, IL-17A+, and Foxp3+ populations were too small to be automatically identified. For these markers, all of the samples were combined into a single file to allow a more robust population identification using a static gate. The measured immunophenotypes were analyzed using receiver operator characteristic (ROC) curves. A cumulative distribution function (CDF) of the area under the curve (AUC) values is illustrated in Figure S4A. The immunophenotypes with an AUC score of higher than 0.9 were selected for analysis using RchyOptimyx [23] (Fig. 3A). To include all of the single-marker immunophenotypes, the CD4+, Foxp3+, IL-17A+, and CD8+ cellLyoplate Flow Cytometry for Biomarker DiscoveryFigure 2. Conventional and lyoplate based flow cytometry platforms have comparable intra- and inter-assay variability. A. Comparison of intra-assay variability between conventional- and lyoplate based- flow cytometry platform (CFP and LFP respectively). Coefficient of variation (CV) was calculated for each sample from experimental triplicates at one time point. Arrows indicate the origin of daughter cell populations. Each dot corresponds to one individual, horizontal bars represent medians. B. Comparison 1315463 of inter-assay variability between CFP and LFP. Cell frequencies obtained from the same leucocyte cone sample run across four different experiments. Percentages of IFN-c+, IL-10+, and IL-17A+ cells were calculated within memory CD4+ T cells (identified as live CD3+CD4+CD45RO+ cells). T regs were identified as live CD3+CD4+CD25highFoxp3+ cells. Average and standard error (ER) are indicated in the two bottom rows. doi:10.1371/journal.pone.0065485.gmedium-throughput processing of the samples, using pre-filled 96 well plates and a plate loader. Moreover, pre-formatted lyoplates, containing the same batch of reagents, can be reliably used through the entire duration of a study and across multiple centres. Therefore, LFP reduces hands-on time, while promoting automation and reagent standardization that are of primary importance in translational and clinical research studies. Our data indicate that lyophilized reagents resulted in more powerful cell stimulation and better marker discrimination, possibly due to improved reagent stability after lyophilisation. In keeping with the increased detection of IFN-c+, IL-10+, Foxp3+ and CD25+ cells, most of the lyophilized antibodies also resulted in increased resolution sensitivity as determined by a higher stain index (SI) on stained PBMC. Of note, tandem dyes PE-Cy5 and APC-H7 showed a decreased SI on stained PBMC compared to cells stained with liquid counterparts, indicating that lyophilisation might have a different impact on different fluorochromes. This aspect should be considered when designing the antibody cocktail to be lyophilized, and a pre-test of the lyophilisation impact onto the specific antibody-fluorochrome combinations should be performed, especially for tandem dye conjugates. If possible,choosing an.