The RNA from the plasma samples was isolated before and after the initiation of ENF treatment using the QIAmp Viral RNA kit (Qiagen). Full-length env/rev genes were amplified through RT-PCR using specific primers as previously described [63]. A subsequent nested PCR was carried out using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) to obtain a fragment corresponding to the gp41 protein (primers MluF2 and RNANestedR corresponding to nucleotides 7726-7747 and 8882-8904 of the HIV HXB2 numbering system, respectively). A fragment corresponding to the gp120 protein was amplified from a NL4-3 plasmid (primers RNANestedF and MluR2 corresponding to nucleotides 5954-5983 and 7727-7747 of the HIV HXB2 numbering system, respectively). The purified gp41 and gp120 products, which overlapped each other in 22 bases, were combined by PCR and purified to obtain the recombinant Envs (gp120 from NL4-3 and gp41 from patients). A directional cloning reaction was performed to insert the fragment into the plasmid expression vector pcDNA.3.1D/V5/His-TOPO (Invitrogen), and several transformed bacterial colonies were selected for each sample. All recombinant plasmids were sequenced using specific primers, the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and an automatic DNA Sequencer (3100 Genetic Analyzer). The sequences were edited (using Sequencher, v4.7, from the Gene Codes Corporation, Ann Arbor, MI and GeneDoc, v2.6, software), and the recombinant plasmids with the required mutations were selected.
Bd Facsdiva Software Download 15
Twenty-four hours post-transfection, cell membrane expression of the Env glycoprotein was assessed by flow cytometry after indirect staining with the anti-gp120 monoclonal antibody 2G12 (4 μg/ml) for 20 min at 37C, followed by staining with phycoerythrin-labeled goat anti-human IgG (RT for 15 min). The cells were washed, fixed in 1% formaldehyde and analyzed by a FACS LSRII flow cytometer. The data were analyzed using FACSDiva software (BD Biosciences). Mock-transfected cells were used as a negative staining control. The percentage of Env-positive cells and the geometric mean fluorescence intensity (geoMFI) of these cells were considered as individual parameters or used to calculate the relative fluorescence intensity (RFI = % of Env-positive cells geoMFI of Env-positive cells), as described previously [50].
Env-induced cytopathic effects were evaluated using a coculture system of Env-expressing HeLa cells as effector cells and labeled primary CD4+ T cells as target cells. The primary CD4+ T cells were stained with the far red cell tracker, DDAO (10 μg/mL), for 1 hour at 37C. Env+ HeLa cells and CD4+/DDAO+ T cells were cocultured for 24 hours in the absence and presence of the inhibitor, JM-2987 (1 μg/mL), and were stained with DiOC6(3) (40 nM) and PI (5 μg/mL) for 1 hour at 37C. Labeled microbeads (Beads Perfect Count, Invitrogen) were added to the stained coculture to quantify the absolute cell loss, and flow cytometry was performed by a FACS LSRII flow cytometer. The data were analyzed by the FACSDiva software (BD Biosciences).
The Fortessa computer uses FACS Diva software to collect data. These files will be uploaded onto the Flow server for the user to then analyze in FlowJo or similar software. You are able to download FlowJo from their website and the core has a USB dongle you can check out free of charge.
Raw data from Illumina sequencers were processed using the exome analysis pipeline (Amelieff). The pipeline performed the following steps. Some sequence data were provided in BAM format and were converted to fastq format with bam2fastx (tophat-2.0.4) [42] for use in the pipeline. First, raw sequence data were cleaned up with QCleaner software (Amelieff): low-quality reads (>20% of the base calls with a Phred score
Finally, SNVs and small indels were annotated using SnpEff v3.2 software. The effects of mutations were categorized into four impact groups: high, moderate, low, and modifier [34]. Results were outputted in VCF format.
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). 2ff7e9595c
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