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Facscanto 2 multicolor flow cytometer

Manufactured by BD
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Sourced in United States

The FACSCANTO II is a multicolor flow cytometer designed for analysis and sorting of cells and particles. It is capable of detecting up to 20 fluorescent parameters simultaneously. The instrument utilizes a blue, red, and violet laser configuration to excite a wide range of fluorophores. The FACSCANTO II provides high-throughput sample processing and data acquisition for a variety of research and clinical applications.

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Lab products found in correlation

2 nbdg, by Thermo Fisher Scientific (1 mentions) Fixation permeabilization solution kit, by BD (1 mentions) Recombinant human il 10, by BioLegend (1 mentions) Diva software, by BD (1 mentions) Annexin 5, by BD (1 mentions) Annexin 5 and 7 aad, by BD (1 mentions) Proteostat aggresome detection kit, by Enzo Life Sciences (1 mentions)

Close spelling variants of this product

FACSCanto II flow cytometer Multicolor flow cytometer FACSCanto flow cytometer FACSCanto II cytometer FACSCanto cytometer FACSCanto II FACSCanto

4 protocols using facscanto 2 multicolor flow cytometer

1

Quantifying Cellular Glucose Uptake

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Glucose uptake assays were done on PBMC stimulated overnight with coated anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) for ex vivo analysis. To study inhibition by specific chemical compounds, PBMCs were stimulated for 40 h with virus-specific peptides. After specific stimulation, PBMCs were washed with PBS 1X to eliminate endogenous glucose and stained for 30 min at 37 ° C with the glucose analog 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose, ThermoFisher; 40 μM) in glucose-free RPMI with 10% dialyzed FBS, and finally stained with the viability probe 7-AAD. Fluorescence generated by the glucose analog, which is proportional to glucose uptake, was measured with a FACSCANTO II multicolor flow cytometer and analyzed with DIVA (BD Biosciences) and FlowJo (Tree Star) softwares.
GLUT 1 assay was performed on PBMCs, after specific stimulation as described above, by using an anti-glucose transporter GLUT1 antibody (Glut1-FITC, R&D Systems). Cells were surface-stained, fixed with the fixation reagent-medium A (Nordic MUbio), then permeabilized with the permeabilization reagent-medium B (Nordic MUbio) and stained with the anti-glucose transporter GLUT1 antibody (R&D system), according to the manufacturer’s instructions, and finally analyzed by flow cytometry.
Barili V., Fisicaro P., Montanini B., Acerbi G., Filippi A., Forleo G., Romualdi C., Ferracin M., Guerrieri F., Pedrazzi G., Boni C., Rossi M., Vecchi A., Penna A., Zecca A., Mori C., Orlandini A., Negri E., Pesci M., Massari M., Missale G., Levrero M., Ottonello S, & Ferrari C. (2020). Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection. Nature Communications, 11, 604.
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2

Phosphoproteomics and Epigenetic Profiling of T-cell Activation

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Phosphoproteins and nuclear factors were measured on PBMC either unstimulated or incubated overnight with coated-anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) or with NS3-/FLU-CMV-EBV-specific peptides in the presence or absence of the antioxidant resveratrol (10 μM).
For anti-phospho-ATM, anti-dimethyl-Histone H3 (Lys9), anti-acetyl-Histone H3 intracellular antibody detection, intracellular staining BD Phosflow™ Fix and Perm/Wash Buffer I (BD) was used, according to the manufacturer’s instructions. For anti-phospho-p38, anti-total p53 and anti-phospho-p53 antibody detection, the intracellular staining Fixation/Permeabilization Solution Kit (Cytofix/Cytoperm BD) was used, according to the manufacturer’s instructions.
Data were collected with a FACSCANTO II multicolor flow cytometer and analyzed with DIVA (BD Biosciences) and FlowJo (Tree Star) softwares.
Barili V., Fisicaro P., Montanini B., Acerbi G., Filippi A., Forleo G., Romualdi C., Ferracin M., Guerrieri F., Pedrazzi G., Boni C., Rossi M., Vecchi A., Penna A., Zecca A., Mori C., Orlandini A., Negri E., Pesci M., Massari M., Missale G., Levrero M., Ottonello S, & Ferrari C. (2020). Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection. Nature Communications, 11, 604.
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3

Measuring IL-10 Effects on Hepatitis B Antigen-Induced Apoptosis

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To measure the effect of IL-10 on Ag-induced apoptosis, peripheral blood mononuclear cells (PBMC) from five HLA-A201+ patients with acute self-limited hepatitis B (concentration of 2×106/ml) were incubated for 1 h at 37 °C with 20 μg/ml of anti-IL-10R Ab (BD Pharmingen), 200 ng/ml of recombinant human IL-10 (BioLegend) or left untreated prior to the addition of Core 18-27 peptide (1 µg/ml) and human IL-2 (100 IU/ml). After a 5 h incubation, cells were extensively washed, stained with Core 18-27 dextramer, anti-CD8 and anti-CD3 mouse Abs for 15 min in the dark, then stained with Annexin V and 7AAD (BD Pharmingen), according to the Annexin V staining protocol (BD Pharmingen). The cells were acquired immediately on a FACSCANTO II multicolor flow cytometer and were analyzed with the DIVA software (BD Biosciences, Immunocytometry Systems, CA, USA).
Fioravanti J., Di Lucia P., Magini D., Moalli F., Boni C., Benechet A.P., Fumagalli V., Inverso D., Vecchi A., Fiocchi A., Wieland S., Purcell R., Ferrari C., Chisari F.V., Guidotti L.G, & Iannacone M. (2017). Effector CD8+ T cell-derived interleukin-10 enhances acute liver immunopathology. Journal of Hepatology, 67(3), 543-548.
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4

Detecting Protein Aggregation in PBMCs

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For the detection of protein aggregates, after overnight PBMC stimulation with or without coated anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml), cells were surface stained and then the ProteoStat Aggresome Detection Kit (Enzo Life Sciences, New York, NY) was used according to the manufacturer’s protocol, before flow cytometry acquisition25 (link). Cells treated with 5 μM of proteasome inhibitor (MG-132) served as positive controls. Aggresome levels were quantified by subtracting ProteoStat median fluorescence intensity (MFI) in the unstimulated from the stimulated samples. Data were collected with a FACSCANTO II multicolor flow cytometer and analyzed with DIVA (BD Biosciences) and FlowJo (Tree Star) softwares.
Barili V., Fisicaro P., Montanini B., Acerbi G., Filippi A., Forleo G., Romualdi C., Ferracin M., Guerrieri F., Pedrazzi G., Boni C., Rossi M., Vecchi A., Penna A., Zecca A., Mori C., Orlandini A., Negri E., Pesci M., Massari M., Missale G., Levrero M., Ottonello S, & Ferrari C. (2020). Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection. Nature Communications, 11, 604.
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