Cyan flow cytometer

Manufactured by Agilent Technologies

Sourced in United States, Denmark

The Cyan flow cytometer is a compact and versatile instrument designed for cell analysis and sorting. It utilizes advanced technology to detect and measure the physical and fluorescent characteristics of individual cells or particles passing through a laser beam. The Cyan flow cytometer provides reliable and accurate data for a wide range of applications in fields such as immunology, stem cell research, and cancer biology.

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Lab products found in correlation

Live dead aqua, by Thermo Fisher Scientific (2 mentions) Cd8 fitc, by Thermo Fisher Scientific (2 mentions) Aqua live dead stain, by Thermo Fisher Scientific (2 mentions) Facscan flow cytometer, by BD (2 mentions) Cd4 cd14 cd16 cd19 tc, by Thermo Fisher Scientific (2 mentions) Facscalibur, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Anti cd8 percp, by BD (1 mentions) Anti ki67 fitc, by BD (1 mentions) Anti cd4 pe cy7, by BD (1 mentions) Anti cxcr3 apc, by BD (1 mentions) Anti cd62l pe, by BD (1 mentions) Anti cd3 pe cy7, by BioLegend (1 mentions) Foxp3 staining buffer, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Merck Group (1 mentions) Anti t bet pe ebio4b10, by Thermo Fisher Scientific (1 mentions) Anti cd127 pe cy7, by BD (1 mentions) Anti cd122 fitc, by BD (1 mentions) Anti cd90.2 pe cy7, by BD (1 mentions) Facs lysing buffer, by BD (1 mentions)

Close spelling variants of this product

CyAn ADP flow cytometer (70 citations) Dako Cyan flow cytometer (10 citations) Cyan cytometer (6 citations) CyAn 7 Color flow cytometer (4 citations)

38 protocols using cyan flow cytometer

Flow Cytometry Phenotyping of CIK Cells

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For human CIK cells, effector (CIK) cells harvested at different time points were washed, stained with various monoclonal antibodies for 2–4 h and analysed by flow cytometry. The following antibodies were used: PE mouse IgG1, k isotype control, PE mouse IgG2a, k isotype control, anti-CD3 FITC, anti-CD56 APC, anti-CCR4 PE, anti-CCR5 PE, anti-CXCR3 PE, anti-CXCR4 PE and anti-CCR7 PE. All the antibodies except anti-CCR7 were purchased from BD Pharmigen (Franklin Lakes, NJ, USA). The CCR7 antibody was purchased from R&D systems. All the antibodies were used according to the manufacturer's directions. Samples were analysed using a CYAN Flow Cytometer (Dako, Carpinteria, CA, USA), and analysis was with FlowJo (Treestar Inc., Ashland, OR, USA). In all cases, DAPI staining was used to determine viability and to gate for live cells.
For mouse CIK cells, the following antibodies were used: mouse anti-CD3 FITC, mouse anti-CD3 PE, mouse anti-CD3 APC, mouse anti-CCR5 FITC and mouse anti-CXCR3 PE. All of the antibodies were purchased from e-Bioscience company (San Diego, CA, USA). All the antibodies were used according to the manufacturer's directions. Samples were analysed using a CYAN Flow Cytometer (Dako) and analysed with FlowJo.

Zou Y., Li F., Hou W., Sampath P., Zhang Y, & Thorne S.H. (2014). Manipulating the expression of chemokine receptors enhances delivery and activity of cytokine-induced killer cells. British Journal of Cancer, 110(8), 1992-1999.

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Multiparameter Flow Cytometry of T-cell Subsets

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Purified CD8⁺ T cells, splenocytes and thymocytes were first stained for surface antigens and then treated with Foxp3 staining buffer set according to the manufacturer's directions (eBioscience). Anti-Eomes AlexaFluor 647 or eFluor 660 (Dan11mag, 1/75), anti-T-bet PE (eBio4B10, 1/100) and anti-CD49d FITC or PE (R1-2, 1/50) antibodies were purchased from eBioscience. Anti-CD8 PercP (53–6.7, 1/50), anti-CXCR3 APC (CXCR3-173, 1/50), anti-CD4 Pe-Cy7 (RM4-5, 1/100), anti-CD62L PE (1/100) or V450 (1/50) (MEL-14), anti-Bcl2 PE (3F11, 1/25), anti-Ki67 FITC (B56, 1/25), anti-CD44 FITC or V450 (IM7, 1/50), anti-CD127 Pe-Cy7 (SB/199, 1/50), anti-CD122 FITC (TM-BETA1, 1/50), anti-NK1.1 FITC (PK136, 1/50), anti-CD90.2 Pe-Cy7 (53-2.1, 1/100) and anti-IFNγ APC or PB or PE (XMG1.21/50) were purchased from BD biosciences. Anti-CD3 Pe-Cy7 (2C11, 1/100) was purchased from Biolegend.
In some experiments, brefeldin A (5 μg ml⁻¹, Sigma) was added in samples for 3 h at 37 °C before intracytoplasmic staining. Blood samples were directly stained for surface antigens and then treated with FACS lysing buffer (BD biosciences) as described in the product data sheet. All samples were fixed with 1% paraformaldehyde in PBS prior to their processing using a Cyan flow cytometer (Dako Cytomation).

Martinet V., Tonon S., Torres D., Azouz A., Nguyen M., Kohler A., Flamand V., Mao C.A., Klein W.H., Leo O, & Goriely S. (2015). Type I interferons regulate eomesodermin expression and the development of unconventional memory CD8+ T cells. Nature Communications, 6, 7089.

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Flow Cytometry Analysis of Murine Immune Cells

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Spleens were homogenized by manual disruption and red cells were lysed as described above. The cells were resuspended at 4°C in FACS buffer (PBS, 5% FBS and 0.1% sodium azide). Antibodies were incubated with cells for 30 min at 4°C at the indicated dilution and washed with FACS buffer before resuspension in fresh FACS buffer. Samples were analyzed on a Cyan flow cytometer (Dako) or an LSR-II flow cytometer (BD). The antibodies used were as follows: anti-Ly6G (48–5931-82; eBioscience; dilution 1/200), anti-CD11c (17–0114-81; eBioscience; dilution 1/200), anti-CD19 (48–0193-82; eBioscience; 1/200), anti CD11b (17–0112-82 or 45–0112-80; eBioscience), anti-CD8a (11–0081-82; eBioscience; 1/400 dilution), anti-CD4 (558107; BD), anti-F4/80 (12–4801-82; eBioscience; 1/200), anti-CD115 (12–1152-82; eBioscience; 1/250), anti CD45.1 (25–0453-82; eBioscience; 1/200), anti-CD45.2 (17–0454-81; eBioscience; 1/200), and anti-Ly6C (53–5932; eBioscience; 1/200).

Thomas D.C., Clare S., Sowerby J.M., Pardo M., Juss J.K., Goulding D.A., van der Weyden L., Storisteanu D., Prakash A., Espéli M., Flint S., Lee J.C., Hoenderdos K., Kane L., Harcourt K., Mukhopadhyay S., Umrania Y., Antrobus R., Nathan J.A., Adams D.J., Bateman A., Choudhary J.S., Lyons P.A., Condliffe A.M., Chilvers E.R., Dougan G, & Smith K.G. (2017). Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity. The Journal of Experimental Medicine, 214(4), 1111-1128.

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Splenocyte Tetramer Staining and Analysis

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Mouse splenocytes were stained for tetramer and pulled down as described previously7 (link). Resulting fractions were then stained for various surface molecules and run on a Dako CyAn flow cytometer. Data were analysed on FlowJo 7.6.5. Tetramer:CD3 ratios were obtained by creating the ratio equation under ‘derived parameters', applying the ratio to the appropriate population and subsequently measuring the population for the gMFI of the derived Tet:CD3 ratio.

White J.T., Cross E.W., Burchill M.A., Danhorn T., McCarter M.D., Rosen H.R., O'Connor B, & Kedl R.M. (2016). Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner. Nature Communications, 7, 11291.

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Phenotyping of PR1-specific CTLs

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Peripheral blood mononuclear cells (PBMC) were prepared by ficoll separation and stained with the following antibody cocktail (Caltag, Burlingame CA): CD8 FITC, CD4/CD14/CD16/CD19 TC, PR1/HLA-A2 or pp65/HLA-A2 tetramer PE ²³ and aqua live/dead stain (Invitrogen, Carlsbad, CA). Events were acquired on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). After gating on live, CD4/CD14/CD16/CD19- negative cells, a CD8+TC- gate was used to identify CTL. PR1-CTL were then identified from this gating schema.
Detailed phenotyping of total CD8 lymphocytes and PR1-CTL at the time of immune response was performed in patients with sufficient sample material. Cells were stained with antibodies as above for tetramer and with antibodies to CD45RA, CCR7, and a dead cell exclusion by aqua live/dead (Invitrogen, Carlsbad, CA). Cells were then acquired on a Cyan flow cytometer (Dako, Ft. Collins, CO). Cell compartments were defined as: Naïve (N=CD45RA+CCR7+), central memory (CM=CD45-CCR7+), effector memory (EM=CD45RA-CCR7-), and terminally differentiated (TD=CD45RA+CCR7-). Statistics were analyzed by paired t test (two-tailed).

Qazilbash M.H., Wieder E., Thall P.F., Wang X., Rios R., Lu S., Kanodia S., Ruisaard K.E., Giralt S.A., Estey E.H., Cortes J., Komanduri K.V., Clise-Dwyer K., Alatrash G., Ma Q., Champlin R.E, & Molldrem J.J. (2016). PR1 Peptide Vaccine Induces Specific Immunity With Clinical Responses In Myeloid Malignancies. Leukemia, 31(3), 697-704.

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Cellular G1 Arrest Assay for Drug Screening

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Example 151

G1 Arrest (Cellular G1 and S-Phase) Assay

For determination of cellular fractions in various stages of the cell cycle following various treatments, HS68 cells (human skin fibroblast cell line (Rb-positive)) were stained with propidium iodide staining solution and run on Dako Cyan Flow Cytometer. The fraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle was determined using FlowJo 7.2 0.2 analysis.

The compounds listed in Table 1 were tested for their ability to arrest HS68 cells at the G1 phase of the cell cycle. From the results of the cellular G1 arrest assay, the range of the inhibitory EC₅₀values necessary for G1 arrest of HS68 cells was from 22 nM to 1500 nM (see column titled “Cellular G1 Arrest EC₅₀” in Table 4).

US10925878B2. HSPC-sparing treatments for RB-positive abnormal cellular proliferation (2021-02-23). G1 Therapeutics, Inc. [US]. Inventors: Jay Copeland Strum [US], John Emerson Bisi [US], Patrick Joseph Roberts [US], Francis Xavier Tavares [US].

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Measuring Calcium Signaling and Cell Motility in CD4+ T Cells

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Preactivated and rested (24 h) primary CD4⁺ cells were transfected with Myc-ACK1 for 24 h and washed with cell loading medium (PBS with 1 mm calcium). Cells were loaded with 1.5 μm Indo-1 dye (Invitrogen) at 37 °C for 30 min. Excess free dye was removed by two washes with cell loading medium. The ratio of bound (405 nm) and free (485 nm) calcium was measured on a CyAn flow cytometer (Dako Cytomation) (48 (link)) upon TCR engagement by hamster anti-CD3 (2C11) plus anti-hamster cross-linking secondary antibody. The recorded data were analyzed and plotted using Summit software (Beckman Coulter).
To perform the motility assay, murine CD4⁺ T cells were activated with plate-bound anti-CD3 and CD28 (2 μg and 1 μg, respectively) for 48 h. Activated cells were then transfected with 2 μg of HA-ACK1 or empty vector control per 1 × 10⁶ cells using the Amaxa Nucleofector Kit (Lonza). Following transfection, cells were maintained in culture without anti-CD3/CD28 for an additional 24 h (i.e. rested). Cells were then tracked on ICAM-1-Fc (2 μg/ml)-coated plates. Alternatively, cells were treated with 2 μm of ACK1 inhibitor (AIM-100) for 30 min before imaging. Images were acquired every 10 s for 20 min. Images were processed using Zeiss LSM510 confocal software and analyzed by Volocity software (Improvision).

Thaker Y.R., Recino A., Raab M., Jabeen A., Wallberg M., Fernandez N, & Rudd C.E. (2017). Activated Cdc42-associated kinase 1 (ACK1) binds the sterile α motif (SAM) domain of the adaptor SLP-76 and phosphorylates proximal tyrosines. The Journal of Biological Chemistry, 292(15), 6281-6290.

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Quantifying Cellular Oxidative Stress

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ROS levels were quantified by staining the cells with MitoSOX Red and CM-H₂DCFDA dyes (both from Invitrogen, Paisley, UK). After 30 minutes incubation with the dyes at 5 μM final concentration, cells were collected and analyzed by flow cytometry using either a FACSCalibur instrument (BD Biosciences, San Jose, CA) or a CyAN flow cytometer (DAKO, Cambridgeshire, UK). Data were analyzed using either CellQuest V or Summit software. Cell incubation with MitoSOX was performed in serum-depleted media.

Funes J.M., Henderson S., Kaufman R., Flanagan J.M., Robson M., Pedley B., Moncada S, & Boshoff C. (2014). Oncogenic transformation of mesenchymal stem cells decreases Nrf2 expression favoring in vivo tumor growth and poorer survival. Molecular Cancer, 13, 20.

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Flow Cytometry Analysis of Lymph Node Cells

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Mice (n = 4 for all groups) were treated with MN patches as described in the IVIS Imaging section. After euthanization at various time points, the draining inguinal lymph nodes were collected and dissociated into single-cell suspensions using frosted slides. Cells were counted, stained with fluorescently labeled antibodies (CD11c-PE, F4/80-APC, B220-eFluor450, PDCA-1-PE-Cy7, CD3-APC-eFluor780, and CD80-PE-Cy7), and analyzed on a Cyan flow cytometer with Summit software (Dako). GC B cells in dLNs were stained with B220-eFluor450, GL7-AlexaFluor647, and CD95-FITC on day 34 post–prime immunization.

Caudill C., Perry J.L., Iliadis K., Tessema A.T., Lee B.J., Mecham B.S., Tian S, & DeSimone J.M. (2021). Transdermal vaccination via 3D-printed microneedles induces potent humoral and cellular immunity. Proceedings of the National Academy of Sciences of the United States of America, 118(39), e2102595118.

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Cell Surface Marker Profiling of PBMCs and TIL

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For cell surface staining, PBMCs and TIL were washed twice in staining buffer (2% w/v fetal bovine serum) and stained for cell surface markers. Cells were incubated with relevant antibodies for 30 min at 4°C in the dark, washed twice and re-suspended in staining buffer. Flow cytometry was performed using a CyAn flow cytometer (Dako, Ft. Collins, CO, USA) and Fortessa cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) and data analyzed using FlowJo software (TreeStar, Inc., Ashland, OR, USA). The acquisition and analysis gates were restricted to the lymphocyte gate based on characteristic properties of the cells in the forward and side scatter.

Kansy B.A., Concha-Benavente F., Srivastava R.M., Jie H.B., Shayan G., Lei Y., Moskovitz J., Moy J., Li J., Brandau S., Lang S., Schmitt N.C., Freeman G.J., Gooding W.E., Clump D.A, & Ferris R.L. (2017). PD-1 status in CD8+ T cells associates with survival and anti-PD-1 therapeutic outcomes in head and neck cancer. Cancer research, 77(22), 6353-6364.

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