Hi!First of all, thanks for this awesome program! I have been finding dificulties in opening the fcs files from my BD accuri C6 plus, and i do not understand the problem given that the last version is able to open any fcs file theoretically. Please find attached one of the FCS files saved from one of my analysis. If there is a format problem, how could i fix it? is there any format converter?Thanks for your help :)
Hi Arantxa,could you describe the problem in more detail, please? I downloaded the FCS file you attached and can open it quite fine in FCSalzyer 0.9.18 - please see the attached screenshot.What exactly does not work for you?Thanks,Sven
Bd Accuri C6 Software Download
Data is collected over 7-decade dynamic range (16 million channels of digital data), making all data available to users as needed. Gating strategies and fluorescence compensation values can be set before, during, or after data collection. After data is collected, the BD Accuri C6 software Zoom function allows for visualization of data at any scale, which allows for precise placement of gates and regions.
Users running samples in tubes will use BD Accuri C6 software (v1.0) for acquisition and analysis of samples. While users running multi-well plates will use IntelliCyt ForeCyt iDM Server Edition software (v3.1).
The ImageStream uses INSPIRE software v4.1 for sample acquisition. Sample analysis is done using either IDEAS software v6.0 (which can be downloaded by users) or FCS Express Plus v5 (which is available to use in the Computer Analysis room in MBB 1.426U).
SPICE - stands for "Simplified Presentation of Incredibly Complex Evaluations". Multicolor flow cytometry experiments generate vast amounts of complex data and require sophisticated software for their evaluation. SPICE is a data-mining software application that analyzes large FlowJo data sets from polychromatic flow cytometry and organizes the normalized data graphically. SPICE enables users to discover potential correlations in their experimental data within complex data sets. Many potential applications for SPICE exist: the software can be used to analyze any multivariate data set for which a series of nominal measurements and a single continuous measurement is available (free).
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Registered users at the facility have access to FlowJo as part of our services at no extra cost. Licenses are activated for one week upon request for remote work. Users will need to download the FlowJo software, and we strongly recommend that you read the FlowJo tutorials and watch the FlowJo webinars before attempting any work.
Aurora users are strongly advised to analyse large quantities of data using SpectroFlo, the software specific for Cytek Aurora. The facility has a powerful computer with SpectroFlo installed that can be used as part of our services at no extra cost. The SpectroFlo workstation can be booked via PPMS (link at the bottom of this page) and is located in the Sir Alexander Fleming Building, 5th floor, Room 532. We strongly recommend that you watch the SpectroFlo tutorials on migrating your experiment instance before analysing your data.
Free flow cytometry software packages are another option if you have experience in coding. Many of these free softwares allow you to write tools that are specific for your analysis. The Purdue University Cytometry Laboratories has a comprehensive list. However please note that the facility staff wont be able to offer assistance on using and coding free software.
Raw flow-cytometry data was extracted from.fcs files exported from BD Accuri C6 software using flowCore (v 2.0.1) Bioconductor package65 and all analysis performed in Rstudio v1.1.463. Rather than using manually placed gates to classify events, unsupervised machine-learning classification algorithms were used. For main flow cytometry plots (Fig. 8) k-means clustering was applied to FL1 height, FL1 area, and Side-Scatter height observations from one replicate using kmeans() function in stats package66 in R, and the clustering solution applied to all the data. Optimal number of clusters was determined empirically using NbClust() function from NBClust package67 in R. To separate P2 and P1 events in permeabilized SG-stained live cells (Fig. 11), a Gaussian Mixture Model was fit to all the data with 2 component distributions using normalmixEM() function in mixtools package68. In all FCM scatter plots, log transformations to base e (natural logarithms) are presented unless otherwise indicated (plots with log transformations to base 2 are used in cases where a doubling of fluorescence is a specific feature of interest).
BD Accuri C6 Plus flow cytometer is a simple, user friendly flow cytometer. Numerous processes are automated, such as daily QC, and the intuitive software interface guides the user through workflows. A wide dynamic range of over 7 decades ensures that all data is available all of the time. Information obtained from the BD Accuri C6 Plus can be reanalyzed at any time if gating or compensation changes are required.
Cell division assay (CDA): CDA was carried out as previously described [8, 9]. The treatment of patient cells was carried out in duplicates or triplicates. The data analysis was carried out using the BD Accuri C6 software as previously described [8]. The T cell proliferation rate in the untreated sample was calculated as the percentage of EdU-positive cells in that sample using the BD Accuri C6 software.
Measurement of γ-H2AX by flow cytometry: H2AX phosphorylation was measured by flow cytometry analysis as previously described [12]. The data analysis was carried out using the BD Accuri C6 software as previously described (12). The γ-H2AX mean fluorescence intensity (MFI) for each time point was calculated by subtracting the γ-H2AX MFI of the non-treated sample harvested at the same time point.
Tet-ON Tap63γ myoblasts were grown in presence or absence of doxycyclin (2µg/µl) for 8, 16 and 24 hours, fixed 30 minutes at 4 ̊C in methanol:aceton (4:1), incubated 20 minutes at 37̊C with RNAse A (20µg/ml) (Sigma, USA) and 20 minutes at room temperature with PI (50µg/ml) (Sigma, USA). 10000 events were acquired using BD FacsCalibur, cell cycle phases and subG1 populations were evaluated by ModFit LTTM software (BD Biosciences).
Whole transcriptome mRNA profiling was performed by Biogazelle (Ghent, Belgium). Briefly 100ng of total RNA was processed using the Quick Amp labeling kit (Agilent), producing Cy3-labelled cRNA. cRNA was hybridized to a SurePrint G3 Mouse GE 8x60K Microarray (Agilent) and microarrays were analyzed using an Agilent microarray scanner and Feature Extraction software. Probe intensities were background subtracted and normalized using quantile normalization. Normalized probe intensities are log2-based. Normalized mRNA expression data and probe annotation are available on request.
Group differences of the same drug treatment with different concentrations were analyzed by one-way ANOVA with Tukey HSD Post Hoc Test using JMP 10 software [21]. No overlapping by the same lower-case letter indicated significant differences.
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