Facs cyan

Manufactured by Beckman Coulter

Sourced in United States

The FACS CyAn is a flow cytometry system designed for analyzing and sorting cells. It is capable of detecting and measuring multiple parameters of individual cells or particles passing through a laser beam. The FACS CyAn is a versatile instrument that can be used for a wide range of applications in various fields, including cell biology, immunology, and stem cell research.

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Lab products found in correlation

Alexa fluor 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Ficoll hypaque, by GE Healthcare (1 mentions) Siglec h, by BioLegend (1 mentions) Tcrγ δ, by BioLegend (1 mentions) Fc block cd16 cd32, by BD (1 mentions) Pdca 1, by BioLegend (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Ifn γ, by BD (1 mentions) Il 17a, by BD (1 mentions) Cd11c clone hl3, by BD (1 mentions) F4 80, by BioLegend (1 mentions) Permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Foxp3 antibody, by Thermo Fisher Scientific (1 mentions) Efluor 780, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Mouse igg1 negative isotype control, by BioLegend (1 mentions) Formaldehyde, by Merck Group (1 mentions) Bio one, by Greiner (1 mentions)

Spelling variants of this product

FACS-Cyan ADP (10 citations) CyAn ADP FACS analyzer (3 citations) CyAn ADP FACS machine (2 citations) FACS CyAn flow cytometer (2 citations) FACS Analyser Cyan ADP (2 citations)

14 protocols using facs cyan

Evaluation of P2X7R Expression in MS

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Peripheral blood mononuclear cells were isolated by a density gradient centrifugation over a Ficoll-Hypaque (Ficoll-Paque PLUS, GE Healthcare) from 20 ml of freshly venous blood from five healthy donors (HD), five relapsing MS patients (MS acute), and five remitting MS patients (MS stable). Cells were stained with pre-titrated Abs, to evaluate the expression of P2X7R within cluster of differentiation 14 (CD14)-positive cells. Briefly, PBMCs (1 × 10⁶) were incubated with P2X7-extracellular epitope antibody (Alomone Labs, Jerusalem, Israel) for 30 min at 4°C. Cells were washed and stained with goat anti-rabbit Alexa Fluor 488-conjugated antibody (Invitrogen, Life Technologies, Monza, MB, Italy), 30 min at 4°C. Cells were washed and stained with anti-CD14 PE (Dako, Aurogene, Rome, Italy) and Live Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen) for 30 min at 4°C.
Monocytes were isolated from PBMCs of HD by using Magnetic Separation with Negative Selection Columns (Miltenyi Biotec, Calderara di Reno, BO, Italy) according to the product manual. Purified monocytes (6 × 10⁶) were cultured in serum-free RPMI 1640 with L-Glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin in 96-well plates.
FACS analysis was performed with FACS CyAn (Beckman Coulter, Pasadena, CA, USA) and with advanced flow cytometry software FlowJo (Tree Star, Ashaland, OR, USA).

Amadio S., Parisi C., Piras E., Fabbrizio P., Apolloni S., Montilli C., Luchetti S., Ruggieri S., Gasperini C., Laghi-Pasini F., Battistini L, & Volonté C. (2017). Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis. Frontiers in Immunology, 8, 1529.

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Comprehensive Immune Cell Profiling

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Ab’s for cell purification and flow cytometry were as follows: CD16/CD32 (Fc Block; BD), TCR Vα2 (BioLegend), CD4 (BioLegend), CD25 (BioLegend), CD69 (BioLegend), CD19 (BioLegend), Foxp3 (BD), IL-17A (BD), IFN-γ (BD), Ki67 (BD), TCR γδ (BioLegend), NK-1.1 (BioLegend), CD49b (BioLegend), CD8a (BioLegend), CD11c (BioLegend), Siglec H (BioLegend), PDCA-1 (BioLegend), B220 (BioLegend), IL-5 (BD), IFN-γ (BD), CD28 (Bioscience), CD3 (eBioscience), and anti–human IgG-Fc (SouthernBiotech). Intracellular cytokine staining was done with the Cytofix/Cytoperm kit (BD). T cell proliferation was assessed by flow cytometry using an anti–human Ki67 staining kit (BD). Foxp3 staining was performed with an eBioscience kit. Flow cytometry was performed with FACS CyAn (Beckman Coulter) and analyzed with FlowJo software.

Sarter K., Leimgruber E., Gobet F., Agrawal V., Dunand-Sauthier I., Barras E., Mastelic-Gavillet B., Kamath A., Fontannaz P., Guéry L., Duraes F.D., Lippens C., Ravn U., Santiago-Raber M.L., Magistrelli G., Fischer N., Siegrist C.A., Hugues S, & Reith W. (2016). Btn2a2, a T cell immunomodulatory molecule coregulated with MHC class II genes. The Journal of Experimental Medicine, 213(2), 177-187.

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Multiparametric Flow Cytometry Analysis

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The frequency of immunological cellular subpopulations in pleural cells was analyzed by flow cytometry. Briefly, cells were stained for 30 min at 4°C with different combinations of the following fluorochrome-conjugated mAb: GR-1 (Clone RB6-8C5), F4/80 (Clone BM8), Ly6C (Clone HK1.4), CD3 (Clone 145-2c11), and CD4 (Clone GK1.5) (BioLegend), and CD11c (Clone HL3) (BD Bioscience), and iNOS (Clone CXNFT) (Cell Signaling technology, eBioscience). After antibody incubation, cells were washed with PBA (phosphate buffered saline containing 0.1% Sodium Azide and 0.2% Albumin bovine). Data were collected using a FACs CyAn flow cytometer (Beckman Coulter, Inc.) and analyzed with FlowJo (Tree Star) software. 100,000 events were acquired per sample.

Chavez-Galan L., Vesin D., Uysal H., Blaser G., Benkhoucha M., Ryffel B., Quesniaux V.F, & Garcia I. (2017). Transmembrane Tumor Necrosis Factor Controls Myeloid-Derived Suppressor Cell Activity via TNF Receptor 2 and Protects from Excessive Inflammation during BCG-Induced Pleurisy. Frontiers in Immunology, 8, 999.

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Multiparametric Flow Cytometry of Immune Cells

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Cells were stained with Ethidium Monoazide bromide (EMA) (Anaspec, USA) for 30 min on ice for live/dead discrimination, and then resuspended in cold FACS buffer (PBS-1 % BSA). Fluorescence-labeled antibodies recognizing CD14, CD11c, HLA-DR, CD80, CD83, CD86, CD4, CD25 and Foxp3 (eBioscience, Germany) were added following manufacturer's instructions for 30 minutes at 4°C in the dark. After incubation, cells were washed and resuspended in FACS buffer before either analysis or staining of intracellular Foxp3 expression. Intracellular staining was performed by incubating the cells with Foxp3 antibody diluted in permeabilization buffer (eBioscience) according to manufacturer's protocol. After 30 minute incubation at room temperature cells were washed, resuspended in FACS buffer and analyzed. Analysis was performed with the FACS CyAn (Beckman Coulter) and the FlowJo software.

Käbisch R., Mejías-Luque R., Gerhard M, & Prinz C. (2014). Involvement of Toll-Like Receptors on Helicobacter pylori-Induced Immunity. PLoS ONE, 9(8), e104804.

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Phosphorylation Analysis of Immune Cells

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For phosphorylation-specific flow cytometry, paraformaldehyde (Merck) was added directly to the culture medium at a final concentration of 1.6% at the end of the stimulation. Cells were fixed for 10 min at room temperature. Subsequently, culture medium was aspirated, resuspended in 100% ice-cold MeOH (Boom) and incubated at −20°C overnight, followed by extensive washing (4×) in PBS with 1% BSA, 0.05% NaN3 (Merck). Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3 (Merck), and 2% human serum (Sanquin), using 1:200 diluted pERK, 1:400 diluted p-p38 Abs, or isotype control (rabbit IgG, Jackson Immuno Research). Cells were stained with primary Ab/isotype control for 45 min on ice, followed by 30 min on ice with a secondary PE-Cy5.5 goat anti-rabbit IgG Ab (Invitrogen, cat# L42018). Data were acquired on an FACSCyan (Beckman Coulter). Isotype controls gave a staining intensity similar as unstimulated moDCs, indicating that blocking conditions were sufficient to avoid unspecific staining (data not shown). Viability was measured using a fixable viability dye eFluor780 (eBioscience), following the manufacturers instructions. Data were analyzed using FlowJo (Treestar).

Søndergaard J.N., van Heeringen S.J., Looman M.W., Tang C., Triantis V., Louche P., Janssen-Megens E.M., Sieuwerts A.M., Martens J.W., Logie C., Stunnenberg H.G., Ansems M, & Adema G.J. (2018). Dendritic Cells Actively Limit Interleukin-10 Production Under Inflammatory Conditions via DC-SCRIPT and Dual-Specificity Phosphatase 4. Frontiers in Immunology, 9, 1420.

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CD89 Expression Quantification by Flow

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10⁵ cells were added per well in 96-wells round bottom plates (Greiner bio-one, Cellstar) and stained with 10 µg/ml mouse anti-CD89 mAbs or a mouse IgG₁ negative isotype control (Biolegend). Subsequently, PE-labeled goat-anti-mouse IgG antibody (Jackson) was added. After final washings, cells were fixed in PBS/0.1% bovine serum albumin (BSA; Fitzgerald)/2%formaldehyde (37%; Sigma). Binding of anti-CD89 antibodies was determined with flow cytometry (FACS Cyan, Beckman Coulter), and expressed as signal-to-noise (S/N) ratios, ie, dividing measured geometric fluorescence intensity of anti-CD89 antibody by measured geometric fluorescence intensity of mouse IgG1 isotype control.

van Delft M.A., Aleyd E., van der Mast R., de Jong N., Boon L., Simons P.J, & van Egmond M. (2023). Antagonizing FcαR1 (CD89) as treatment in IgA-mediated chronic inflammation and autoimmunity. Frontiers in Immunology, 14, 1118539.

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Lectin Staining for Flow Cytometry

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For lectin staining, the cells were first washed with 1 × carbo-free blocking solution. Next, the cells were incubated for 45 min at 4 °C with biotinylated MALII (5 µg/ml), SNA-I (1 µg/ml), or PNA (5 µg/ml) in 1 × carbo-free blocking solution supplemented with 1 mM MgCl₂ and 1 mM CaCl₂ (both from Merck). After incubation, the cells were washed with PBA (1 × PBS, 1% bovine serum albumin, 0.02% sodium azide), and incubated with Streptavidin-PE for 20 min at 4 °C. For antibody staining, the cells are harvested, washed in 1 × PBS and stained with eFluor 780 or 450 viability dye according to the manufacturer’s instructions. Fc receptors were blocked with anti-CD16/CD32 (2.4G2, BD) in PBA for 10 min at 4 °C. Afterwards, the cells were stained with fluorescent antibodies in PBA at 4 °C for 20 min, and washed three times with PBA. Samples were acquired with a CytoFLEX LX Flow Cytometer (Beckman Coulter), BD FACSVerse™ flow cytometer (BD Bioscience), Gallios (Beckman Coulter) or FACS Cyan (Beckman Coulter), respectively. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).

Balneger N., Cornelissen L.A., Wassink M., Moons S.J., Boltje T.J., Bar-Ephraim Y.E., Das K.K., Søndergaard J.N., Büll C, & Adema G.J. (2022). Sialic acid blockade in dendritic cells enhances CD8+ T cell responses by facilitating high-avidity interactions. Cellular and Molecular Life Sciences, 79(2), 98.

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Flow Cytometry Analysis of Transduced Cells

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For flow cytometry analysis, micromasses were melted on ice for 2 h, 48 h after transduction with lentiviral PGK-GFP. The cell suspension was incubated with antibodies 11-Fibrau and subsequently with donkey-anti-mouse conjugated to Alexa Fluor 568 (1:100 for 30 minutes) (A-10037, Thermo Fisher Scientific), or cell suspensions were incubated with mouse-anti-human CD68, conjugated to PE (1:20 for 60 minutes) (12-0689, eBioscience, San Diego, CA, USA). The flow cytometry was performed on the FACS Cyan (Beckman Coulter, Woerden, The Netherlands) using the 488 nm laser at the FL1, FL3 and FL7 channels for GFP, 11-Fibrau and CD68 respectively.

Broeren M.G., de Vries M., Bennink M.B., Arntz O.J., van Lent P.L., van der Kraan P.M., van den Berg W.B., van den Hoogen F.H., Koenders M.I, & van de Loo F.A. (2016). Suppression of the inflammatory response by disease-inducible interleukin-10 gene therapy in a three-dimensional micromass model of the human synovial membrane. Arthritis Research & Therapy, 18, 186.

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H. pylori Infection of Gastric Cancer Cells

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Human gastric cancer cells were seeded in 96-well plates (1.5x10⁵ cells per well). Different H. pylori strains were labeled by cell tracer CFDA-SE (Life Technologies, USA) in PBS at 37°C for 30min. After washing three times with PBS, labeled H. pylori were added at MOI 10 to the cells and incubated for 1h at 37°C. Samples were washed with PBS three times and fixed with 100μL 4% paraformaldehyde (PFA) for 5min. Finally, the samples were resuspended with 200μL FACS buffer and analyzed by flow cytometry. Analysis was performed with a FACS CyAn (Beckman Coulter) and the FlowJo software.

Zhong Y., Anderl F., Kruse T., Schindele F., Jagusztyn-Krynicka E.K., Fischer W., Gerhard M, & Mejías-Luque R. (2016). Helicobacter pylori HP0231 Influences Bacterial Virulence and Is Essential for Gastric Colonization. PLoS ONE, 11(5), e0154643.

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CCR5 Knockdown Assay in HeLa Cells

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All cell lines were cultured in high-glucose DMEM (Sigma) supplemented with 10% fetal calf serum, 1% penicillin, 1% streptomycin, and 1% L-glutamine. For each knockdown assay, cells were analyzed at least 5 days after transduction. For CCR5 knockdown studies, a subclone of HeLa-derived TZMbl cells (AIDS Repository, Germantown, MD, USA), expressing high levels of human CCR5, named here HeLa R5, was used. For GFP knockdown, the same cells were used after GFP transduction at one copy of the vector and sorting of the GFP-positive cells. CCR5 expression was detected using an anti-human CCR5-allophycocyanin (APC) antibody (Cat. 550856; BD Pharmingen) and flow cytometry analysis using fluorescence-activated cell sorting (FACS) Cyan (Beckman Coulter). GFP expression was assessed on the same flow cytometer using GFP fluorescence median. In brief, HeLa cells were transduced at 0.2 MOI with the miRGE-based knockdown vector to avoid the presence of a high copy number of the vector per cell and to obtain comparable conditions. GFP or CCR5 expression was compared between the transduced and the remaining untransduced population of cells and expressed as a percentage of CCR5 expression relative to the untransduced population.

Rousset F., Salmon P., Bredl S., Cherpin O., Coelho M., Myburgh R., Alessandrini M., Perny M., Roccio M., Speck R.F., Senn P, & Krause K.H. (2018). Optimizing Synthetic miRNA Minigene Architecture for Efficient miRNA Hairpin Concatenation and Multi-target Gene Knockdown. Molecular Therapy. Nucleic Acids, 14, 351-363.

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