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Fascalibur flow cytometry

Manufactured by BD
Sourced in United States

The FASCalibur is a flow cytometer designed for the analysis of cells and other particles. It is capable of detecting and measuring multiple parameters of individual cells flowing in a fluid stream. The FASCalibur provides accurate and reliable data on cell size, granularity, and fluorescence properties.

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3 protocols using fascalibur flow cytometry

1

Flow Cytometry Analysis of Regulatory T Cells

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Splenocytes were incubated with 2.4G to block FcRs and then incubated with optimal concentration of fluorochrome-labeled mAbs for 30 minutes at 4°C in dark [10 (link)]. Cells were washed three times and resuspended by FCM buffer (PBS with 0.1% BSA and 0.1%NaN3). At least ten thousand cells were assayed using a FASCalibur flow cytometry (Becton Dickinson, CA), and data were analyzed with CellQuest software (Becton Dickinson, Mountain View, CA). In some experiments, non-viable cells were excluded using the vital nucleic acid stain propidium iodide (PI).
For the staining of intracellular Foxp3, cells were incubated with Cy-chrome-labeled anti-CD4 and FITC-labeled anti-CD25 mAbs first [11 (link)]. Then these cells were fixed after washing and stained with anti-mouse Foxp3 mAb, on the basis of the instruction provided by the company (eBioscience, San Diego, CA).
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2

Flow Cytometric Analysis of SMO and GLI1

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The expressions of SMO and GLI1 were also evaluated by Flow cytometry. A2780 and A2780/DDP cells were washed once with PBS and divided to three aliquots with a density of 106 cells/ml. Cells were incubated respectively with PBS, SMO antibody (1:200; ab72130), or GLI1 antibody (1:50; sc-20687) for 30 min at room temperature, and then stained with Goat anti-Rabbit IgG FITC (Sigma-Aldrich, St. Louis, MO, USA) for 20 min in the dark. Incubation was stopped by adding 500 µl phosphate-buffered saline, followed by washing with PBS for 3 times. At least 10,000 cells were assayed by FCM using a FASCalibur flow cytometry (Becton Dickinson, Mountain View, CA, USA). The expressions of SMO and GLI1 were analyzed with CellQuest software (Becton Dickinson).
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3

Asthmatic Mice Lymphocyte Assay

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CS-BCG-PSN-OVA, BCG-PSN, OVA, and BCG-PSN-OVA were cultured with spleen-derived lymphocytes of asthmatic mice and then co-cultured with OVA. Cells were firstly incubated with antigen-presenting cell-labeled anti-CD4 and phycoerythrin (PE)-labeled anti-CD25 monoclonal antibodies (mAbs). Having been washed, cells were fixed and stained with Alexa Fluor 488-conjugated anti-mouse Foxp3 mAbs or negative control mAbs. The cells were washed 3 times and resuspended in FCM buffer (PBS, 0.1% BSA and 0.1% NaN3). FASCalibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) was used, and the data were analyzed using Cell-Quest software. Firstly, FSC/SSC was set as a gate to isolate lymphocytes. The CD4/CD25 gate was used. The percentage of Foxp3-stained CD4+CD25+ T cells was determined by the cell percentage of T cells stained with indicated anti-mouse mAbs minus that of the cells nonspecifically stained with negative control mAbs in the same dot-plot region.
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