Cyan cytofluorimeter

Manufactured by Agilent Technologies

Sourced in Italy

The CyAn cytofluorimeter is a flow cytometry instrument designed for the detection and analysis of fluorescent-labeled cells. It provides high-speed data acquisition and analysis capabilities for a wide range of applications in life science research.

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Lab products found in correlation

Anti cd4 pe, by Miltenyi Biotec (1 mentions) Anti cd8 fitc, by Miltenyi Biotec (1 mentions) Anti cd3 apc, by Miltenyi Biotec (1 mentions) L mimosine, by Merck Group (1 mentions) Propidium iodide rnase a solution, by Merck Group (1 mentions)

3 protocols using cyan cytofluorimeter

Identification of mouse CSC by c-Kit

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Bone marrow cells were re-suspended in FACS buffer (PBS/2% FBS/2 mM EDTA) and stained for 1 h at 4 °C with rat anti-c-Kit CD117 Alexa Fluor 647 (Invitrogen: RM6221, Thermo Fisher Scientific-Life technologies, Waltham, MA, USA) antibody. Dead cells were stained with 7 Amino-Actinomycin D dye (Sigma Aldrich).
White blood cells were stained in 100 μl of FACS buffer with rat anti-CD11b PE-Cy7 (Pharmingen: 25-0112, Gurgaon, Haryana, India) and rat anti-GR-1 APC antibodies (Pharmingen: 17-5931) at 4 °C for 1 h. Dead cells were stained with Sytox-Blue dye. Analysis was performed with CyAn cytofluorimeter (Dako, Milano, Italy).
Isolated CSC analysis was performed on FACSCanto II with FlowJo software to identify c-Kit expression by mouse monoclonal APC-conjugated anti-c-Kit antibody (Miltenyi). Appropriate labeled isotype controls were used to define the specific gates.

Di Siena S., Gimmelli R., Nori S.L., Barbagallo F., Campolo F., Dolci S., Rossi P., Venneri M.A., Giannetta E., Gianfrilli D., Feigenbaum L., Lenzi A., Naro F., Cianflone E., Mancuso T., Torella D., Isidori A.M, & Pellegrini M. (2016). Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury. Cell Death & Disease, 7(7), e2317-.

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Multiparametric Analysis of Thymocytes

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Single-cell suspensions of thymocytes were stained for 20 min with anti-CD4-PE, anti-CD8-FITC, or anti-TCR-β-APC antibodies (1:50; Miltenyi Biotec, Bologna, Italy) at 4 °C. Dead cells were stained with SYTOX® Blue (Thermo Fisher Scientific-Life Technologies, Waltham, MA, USA). Analysis was performed with CyAn cytofluorimeter (Dako, Milan, Italy) and analyzed using FlowJo software (version 7.2.4). Blood was collected from mouse eyes 1 week before the sacrifice and cells stained with anti-CD45-eFluor450 antibody (1:100; eBioscence, San Diego, CA, USA) to exclude red cells and with anti-CD3-APC, anti-CD4-PE, and anti-CD8-FITC antibodies (1:50; Miltenyi Biotec) to detect mature T cells.

Di Siena S., Campolo F., Gimmelli R., Di Pietro C., Marazziti D., Dolci S., Lenzi A., Nussenzweig A, & Pellegrini M. (2018). Atm reactivation reverses ataxia telangiectasia phenotypes in vivo. Cell Death & Disease, 9(3), 314.

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Cell Cycle Synchronization and Analysis

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HL-1 and NIH3T3 cells were initially plated at a density of 5 x 10 5 cells/well in 3.5 cm dishes. Next day cells were transfected with PDE5A isoforms GFP-tagged vectors. Twenty four hours later, cells were synchronized with 0.5 mM L-Mimosine (Sigma-Aldrich, CA, USA). After 16 hrs, cells were washed with PBS and incubated in fresh culture media. At various times after release from the L-Mimosine block the cells were treated with trypsin, harvested, and centrifuged at 2000 g for 5 min.
The pellets were washed with PBS and then centrifuged again. Cells were fixed with 1% w/v paraformaldehyde for 1hr at 4°C, then centrifuged for 5 min at 300 g; supernatant was removed by aspiration and pellets washed with PBS. For cell membrane solubilization, 1 ml 70% v/v ethanol was added to the cell pellet, and cell suspension incubated overnight at 4°C. Fixed cells were washed with PBS, centrifuged and incubated for 3 hr at room temperature with propidium iodide/RNAse A solution (PI, 50 μg/mL, Sigma-Aldrich, CA, USA), ribonuclease (1 mg/ml Sigma-Aldrich, CA, USA). The cell cycle distribution and DNA content were measured using a CyAn cytofluorimeter (Dako). 5 x 10 3 cells were counted for each sample. The percentage of cells in different phases of the cell cycle was analyzed by FlowJo (TreeStar, Ashland, OR, USA).

Campolo F., Zevini A., Cardarelli S., Monaco L., Barbagallo F., Pellegrini M., Cornacchione M., Di Grazia A., De Arcangelis V., Gianfrilli D., Giorgi M., Lenzi A., Isidori A.M, & Naro F. (2018). Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line. Journal of cellular physiology, 233(1).

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