Bd Accuri C6 Software Download ~UPD~
Bd Accuri C6 Software Download
Imaging Flow Cytometry (ImageStreamX; Amnis) is a powerful tool that obtains high-content, wide field fluorescence imaging of cells expressing fluorescent reporters using the available spatial information offered by the fluorescence microscopy device. Like any other flow cytometry, the resolution is limited by the number of events generated by the flow cell. In principle, imaging allows a high-throughput measurement of thousands of cells. For living cell imaging using the Amnis Imaging Flow Cytometer, cells are re-suspended in 5µl of cell staining solution and this is combined with 25µl of a molten mixture of agarose and phenol red that will solidify to form a bed for the cells. The cells are then layered on the imaging cassette using a micropipette and visualized after adding the proprietary Live Cell Imaging (LCI) Fix and Perm reagent that causes an approximately 5min “fixed and permeabilized” period. At the conclusion of the fixation, the cells are observed by real-time imaging of fluorescence by the stream. Following extensive image analysis, the software provides a.csv file with an array of quantitative measures of the data for each cell. Two options are provided: measures are normalized to a reference population that is used to compensate for photorefractive non-uniformity between channel and across the images (these were done on all samples in experiment) or the measures are normalized against the mean of the images themselves. Though highly scalable, the throughput of the imaging flow cytometer is limited by the capture of any 5µm wide plane of the image (frame) at approximately 60 cells/s. Thus the instrument is not appropriate for studying rare populations.
After 24h treatments, cells were collected and permeabilized using 0.5% saponin (Sigma Aldrich, MO, USA) for 10min. After this period, cells were washed, followed by incubation with eFluor 660 fixable viability dye (ThermoFisher, MA, USA) for 20min. Next, the sample was washed with 1% BSA and then stained with anti-CD4 (APC) (BD Biosciences, CA, USA) and anti-IFN-γ (PE) antibodies (BD Biosciences) at 4°C, in the dark, for 30min, followed by incubation with the permeabilizing agent as described above. Next, cells were washed with 1% BSA and then incubated with anti-IL-17 (PE-cy7) antibodies (BD Biosciences) at 4°C, in the dark, for 30min. Finally, cells were incubated with 7-AAD (Molecular Probes, CA, USA) at room temperature, for 20min. Flow cytometry was performed using FACS Canto II flow cytometer (BD Biosciences). Firstly, the events were gated in order to avoid cell doublets. The percentage of CD4+/IFN-γ+ and CD4+/IL-17+ were determined and the results were presented as bar-charts using the BD Accuri C6 software.
Gating strategy for determination of cytokine producing cells in the Peyers patch (PP) following oral delivery of F-buffer, compared to M-buffer for 8 days. After the 8 days treatment period, Peyer’s patch (PP) was homogenized and cells from the PP were isolated. The cells were stained with FITC-conjugated anti-CD3 and APC-labeled anti-CD4 antibodies. In addition, the cells were also stained with PE-labeled anti-Foxp3 (clone PCH101) and PE-labeled anti-IL-17 (clone eBio64DEC17). The respective isotype controls for the fluorochrome-conjugated antibodies were also stained. Flow cytometry was performed using FACS Canto II flow cytometer (BD Biosciences). Firstly, the events were gated in order to avoid cell doublets. The absolute number of Treg cells was determined using the BD Accuri C6 software. Finally, the percentage of IL-17+ and Foxp3+ cells were determined.