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Cyan flow cytometer

Manufactured by Beckman Coulter
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Sourced in United States, United Kingdom, Japan

The CyAn flow cytometer is an analytical instrument used for the measurement and analysis of particles, cells, or other microscopic objects in a fluid stream. It utilizes principles of light scattering, fluorescence, and impedance to provide quantitative and qualitative data on various properties of the measured samples.

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Lab products found in correlation

Summit software, by Beckman Coulter (10 mentions) Cytofix cytoperm kit, by BD (4 mentions) Ionomycin, by Merck Group (3 mentions) Golgistop, by BD (3 mentions) Summit v4, by Beckman Coulter (3 mentions) Propidium iodide, by Merck Group (3 mentions) Multicycle av software, by Phoenix Pharmaceuticals (3 mentions) Bio plex pro mouse cytokine assay, by Bio-Rad (2 mentions) Duoset elisa kit, by R&D Systems (2 mentions) Summit 5, by Beckman Coulter (2 mentions) Anti mouse cd4, by BD (2 mentions) Anti cd16 cd32, by BD (2 mentions) Live dead cell viability kit, by Thermo Fisher Scientific (2 mentions) Live dead cell viability kits, by BD (2 mentions) Il 27p28, by BD (2 mentions) Fixation and permeabilization solution kit, by BD (2 mentions) Hepes buffer, by Merck Group (2 mentions) Collagenase type 2, by BD (2 mentions) Collagenase type 2, by Merck Group (2 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

CyAn ADP flow cytometer (214 citations) Cyan cytometer (13 citations) CyAn ADP Analyzer flow cytometer (11 citations) Cyan ADP 9-color flow cytometer (5 citations) CyAn ADP High-Performance Flow Cytometer (5 citations) Cyan ADP High Performance Research Flow Cytometer (4 citations) CyAn Analyser flow cytometer (4 citations) CyAn ADP 9C flow cytometer (3 citations) CyAn ADP Lx 9 color flow cytometer (3 citations) CyAn II flow cytometer (3 citations)

103 protocols using cyan flow cytometer

1

Flow Cytometric Analysis of Co-stimulatory Molecules

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Cells were harvested with cold PBS at indicated time points, washed twice in PBS and stained with fixable viability dye (eBioscience) for 30 minutes on ice. Subsequently, cells were stained as described [33 (link)] with conjugated antibodies directed against cell surface markers or corresponding isotypes. CD80-PeCy7, CD86-PeCy7, PD-L1-PE and PD-L2-PE were all from BD Pharmingen; ICOS-L-PerCP was from R&D systems and B7-H3-Alexa488 and B7-H4-Alexa488 were from AbD Serotec. In Min6 co-cultures, expression of co-stimulatory molecules was analyzed on CD11c-APChigh (BD) or BDCA3-APC (Miltenyi) expressing mDCs for BDCA1 and BDCA3 mDCs, respectively. Cells were analyzed on a CyAn Flow cytometer (Beckman Coulter) and data was analyzed using FlowJo software, gated on living cells. Pulse width analysis was included to exclude doublets and only analyze single cells. An example of the gating strategies used is shown in S1 Fig.
Schulte B.M., Gielen P.R., Kers-Rebel E.D., Schreibelt G., van Kuppeveld F.J, & Adema G.J. (2015). Enterovirus-Infected β-Cells Induce Distinct Response Patterns in BDCA1+ and BDCA3+ Human Dendritic Cells. PLoS ONE, 10(3), e0121670.
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2

Multiplex Cytokine Profiling of Mammary Tumors

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Multiplex quantification of cytokines and chemokines in mammary glands and tumors was performed using the premixed 24-plex Bio-Plex Pro Mouse Cytokine Assay (Bio-Rad) according to manufacturer’s recommendations. Proteins lysates were prepared as previously described31 . Unsupervised clustering was performed on normalized, median centered data then converted to a heat-map using Genesis software. For IL17A and G-CSF serum levels, BD Cytometric Bead Arrays were used as directed and analyzed on a Cyan flow cytometer with Summit software (Beckman Coulter). Data analyses were performed using FlowJo Software version 9.7.1. For TGFβ1, a DuoSet ELISA kit was purchased from R&D Systems and performed according to the manufacturer’s instructions.
Coffelt S.B., Kersten K., Doornebal C.W., Weiden J., Vrijland K., Hau C.S., Verstegen N.J., Ciampricotti M., Hawinkels L.J., Jonkers J, & de Visser K.E. (2015). IL17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis. Nature, 522(7556), 345-348.
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3

Cell Cycle Analysis by Flow Cytometry

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DNA content was assayed using propidium iodide. Cells were harvested, washed and fixed in 70% ethanol. Cells were permeabolized with 0.5% Triton X-100 and stained with propidium iodide. Stained cells were quantitated on the CyAN flow cytometer (Beckman Coulter). Distribution of cells in each stage of the cell cycle was performed using ModFit (Verity Software House).
Stratford J.K., Yan F., Hill R.A., Major M.B., Graves L.M., Der C.J, & Yeh J.J. (2017). Genetic and pharmacological inhibition of TTK impairs pancreatic cancer cell line growth by inducing lethal chromosomal instability. PLoS ONE, 12(4), e0174863.
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4

Primary Mouse Lung Endothelial Cell Isolation

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Primary mouse lung EC were isolated from control Ergfl/fl and ErgcEC-het mice as described (Reynolds et al., 2002 (link)). Rat APC-CD31, anti-ICAM-2, and anti-rat PE antibodies (all BD Biosciences) were used to assess the EC purity by flow cytometric analysis using a Cyan flow cytometer (Beckman Coulter).
Birdsey G.M., Shah A.V., Dufton N., Reynolds L.E., Osuna Almagro L., Yang Y., Aspalter I.M., Khan S.T., Mason J.C., Dejana E., Göttgens B., Hodivala-Dilke K., Gerhardt H., Adams R.H, & Randi A.M. (2015). The Endothelial Transcription Factor ERG Promotes Vascular Stability and Growth through Wnt/β-Catenin Signaling. Developmental Cell, 32(1), 82-96.
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5

Flow Cytometric Analysis of T Cells

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Flow cytometric analysis was performed on blood and liver-infiltrating T cells using a Cyan flow cytometer (Beckman Coulter, Bucks, United Kingdom), and analyzed using FlowJo (version 9; Treestar Inc, Ashland, OR) (see the Supplementary Materials and Methods section and Supplementary Table 1).
Liaskou E., Jeffery L.E., Trivedi P.J., Reynolds G.M., Suresh S., Bruns T., Adams D.H., Sansom D.M, & Hirschfield G.M. (2014). Loss of CD28 Expression by Liver-Infiltrating T Cells Contributes to Pathogenesis of Primary Sclerosing Cholangitis. Gastroenterology, 147(1), 221-232.e7.
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6

Immune Cell Profiling by Flow Cytometry

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The antibodies we used in this study were anti-mouse CD4, CD19, CD45, B220, CD3, F4/80, CD11c, CD11b (BD Bioscience), IL-10, IL-17, IFNγ, IL-27p28 and Foxp3 (eBioscience), eBi3 (R&D systems) and LIVE/DEAD Cell Viability Kits (Invitrogen). MLN or colonic LP cells were collected and incubated for 15 minutes at 4°C with anti-CD16/CD32 (BD Bioscience) and then for 20 minutes at 4°C with antibodies for cell surface and LIVE/DEAD Cell Viability Kits to evaluate the cell phenotype and CD4+ T cell/B cell reconstitution. Enumeration of cells expressing IL-10, IL-17, IFNγ, IL-27p28, Ebi3 and Foxp3 was performed by intracellular staining with a Fixation and Permeabilization Solution Kit (BD Bioscience) according to the manufacturer's instructions. For intracellular cytokine staining, 100ng/ml PMA, 1µg/ml Ionomycin (Sigma-Aldrich) and Golgi stop (BD Biosciences) were added into the medium during the last 4 hours of the culture period. Cells were washed and then analyzed on a CyAn flow cytometer (Beckman Coulter, Brea, CA, USA). Proper isotype antibodies were used as a control and gated live CD45+ cells were analyzed with Summit 5.2 software (Beckman Coulter)
Mishima Y., Liu B., Hansen J.J, & Sartor R.B. (2015). Resident Bacteria-Stimulated Interleukin-10-Secreting B Cells Ameliorate T-Cell-Mediated Colitis by Inducing T-Regulatory-1 Cells That Require Interleukin-27 Signaling. Cellular and Molecular Gastroenterology and Hepatology, 1(3), 295-310.
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7

Detection of Caspase-1 Activation in Cells

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To detect caspase-1 activation, cells were activated by LPS and nigericin or EPEC infection, followed by labelling with FAM-YVAD-FMK caspase-1 (FAM-FLICA, Immunochemistry, Bloomington, IN, USA) for 30 min. PI (0.5% (v/v)) was added for the last 5 min of incubation to label dead cells. The presence of active caspase-1 and incorporation of PI was analyzed by Cyan flow cytometer (Beckman Coulter) and the FlowJo software. The amount of IL-1β released in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Lille, France). IL-1 receptor expression on BMDC was determined by staining with APC anti-mouse CD121a (IL-1R, JAMA-147; BioLegend) by flow cytometry. Cytotoxicity induced was evaluated by LDH assay using the CytoTox96 LDH-Release Kit (Promega), as described by the manufacturer’s protocols.
Lee P.P., Lobato-Márquez D., Pramanik N., Sirianni A., Daza-Cajigal V., Rivers E., Cavazza A., Bouma G., Moulding D., Hultenby K., Westerberg L.S., Hollinshead M., Lau Y.L., Burns S.O., Mostowy S., Bajaj-Elliott M, & Thrasher A.J. (2017). Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells. Nature Communications, 8, 1576.
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8

CD4+ T Cell Migration Assay

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PBMCs from the healthy controls and SLE patients were sorted by negative selection using the CD4 + T Cell Isolation Kit II (Miltenyi Biotech). A transwell system with a pore size of 0.5 mm (Corning, Lowell, MA) was used for migration assay. In the bottom compartment, IL-15 (R&D Systems) was added at increasing concentrations of 0.1ng/ml, 1ng/ml and 10 ng/ml. 5 × 105 CD4 + T cells were seeded in each insert. After 5 h incubation, the total number of migrating cells was counted on a Cyan flow cytometer (Beckman Coulter), and the percentage of both CD4 + CD28− T and CD4 + CD28 + T cells was determined. The chemotactic index (CI) was calculated as previously described [18 (link)], by dividing the number of cells that migrated in the presence of IL-15 by the number of cells that migrated in the absence of IL-15.
Zhang T., Liu X., Zhao Y., Xu X., Liu Y, & Wu X. (2022). Excessive IL-15 promotes cytotoxic CD4 + CD28− T cell-mediated renal injury in lupus nephritis. Immunity & Ageing : I & A, 19, 50.
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9

Assessing T cell-endothelial interaction and apoptosis

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GEnCs were purchased from Siencell (CA, USA). CD28− and CD28 + T cells were sorted from purified CD4 + T cells using the CD28 Microbead kit II (Miltenyi Biotech, Kölle, Germany). The purity of the sorted CD28- and CD28 + T cells ranged from 90 to 95%. Cells were stimulated with IL-15 (50ng/ml) for 24 h and then incubated with GEnCs in 96-well plates for 6 h. One well per patient was incubated with either 1 µg/ml anti-NKG2D antibody or an isotype control (BD Biosciences). Cells were stained for activated caspase 3 (as a marker of apoptosis) using anti-caspase 3-PE (BD Biosciences) and also stained with anti-IFN-gamma-Pacific Blue following staining for surface markers using anti-CD31-APC (GEnC) and anti-CD28-PE (Biolegend). Cell apoptosis rates and IFN-γ secretion were analyzed by flow cytometry using a Cyan flow cytometer (Beckman Coulter).
Zhang T., Liu X., Zhao Y., Xu X., Liu Y, & Wu X. (2022). Excessive IL-15 promotes cytotoxic CD4 + CD28− T cell-mediated renal injury in lupus nephritis. Immunity & Ageing : I & A, 19, 50.
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10

Dissociation of Organoids for Flow Cytometry

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Organoids were dissociated for flow cytometry analysis using collagenase type II (Sigma-Aldrich, cat. #C6885) resuspended in HEPES buffer (Sigma-Aldrich, cat. #H0887) at a concentration of 20 mg/mL. Samples were collected by gravitation in a 15-mL Falcon tube and washed 2× in PBS and then resuspended in collagenase type II. For dissociation, samples were incubated at 37°C for 5 minutes before trituration and a further 5-minute incubation. The dissociation reaction was stopped through the addition of PBS supplemented with FBS. Ten organoids were dissociated per flow cytometry experiment. Analysis was performed using either a cyan flow cytometer (Beckman Coulter) or an Attune NxT. Single color stained controls and fluorescence-minus-one controls were used for all experiments using antibodies as listed in Supplementary Table S5.
Khan A.O., Rodriguez-Romera A., Reyat J.S., Olijnik A.A., Colombo M., Wang G., Wen W.X., Sousos N., Murphy L.C., Grygielska B., Perrella G., Mahony C.B., Ling R.E., Elliott N.E., Karali C.S., Stone A.P., Kemble S., Cutler E.A., Fielding A.K., Croft A.P., Bassett D., Poologasundarampillai G., Roy A., Gooding S., Rayes J., Machlus K.R, & Psaila B. (2022). Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies. Cancer Discovery, 13(2), 364-385.
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