Cyan adp

Manufactured by Beckman Coulter

Sourced in United States, United Kingdom, Italy, Germany

The CyAn ADP is a flow cytometry instrument designed for analysis and sorting of cells, particles, and molecules. It provides high-speed data acquisition and advanced multi-parameter detection capabilities. The CyAn ADP is a versatile tool for various applications in life science research and clinical diagnostics.

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Lab products found in correlation

Propidium iodide, by Merck Group (14 mentions) Lsrfortessa, by BD (9 mentions) Ionomycin, by Merck Group (7 mentions) Moflo xdp, by Beckman Coulter (6 mentions) Bovine serum albumin (bsa), by Merck Group (6 mentions) Summit 4, by Beckman Coulter (6 mentions) Facsaria 3, by BD (5 mentions) Fortessa, by BD (5 mentions) Modfit lt software, by Verity Software House (4 mentions) Collagenase d, by Roche (4 mentions) Cytofix cytoperm, by BD (4 mentions) Prism 5, by GraphPad (4 mentions) Summit software, by Beckman Coulter (4 mentions) Ack lysis buffer, by Thermo Fisher Scientific (4 mentions) Facscanto 2, by BD (4 mentions) Fcs express 5, by De Novo Software (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Sytox green, by Thermo Fisher Scientific (3 mentions) Dnase 1, by Roche (3 mentions) Golgistop, by BD (3 mentions)

Spelling variants of this product

CyAn ADP flow cytometer (214 citations) CyAn ADP cytometer (33 citations) Cyan ADP 9-color (7 citations) Cyan ADP 9-color flow cytometer (5 citations) CyAn ADP 7 (3 citations) CyAn ADP FACS analyzer (3 citations) CyAn™ ADP High-speed Analyzer (3 citations) Cyan ADP Flow cytometry analyzer (2 citations) Cyan ADP 9 colors cytometer (2 citations) CyAn™ ADP LX nine‐colour analyser (2 citations)

316 protocols using cyan adp

Quantifying Cellular ROS and Apoptosis

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To evaluate ROS levels, cells were incubated for 4 h with ATO, ASC or the combination of the two. The Abcam's ROS assay kit “ab113851” (Abcam, Cambridge, UK) uses the cell permeant reagent 2′,7′–dichlorofluorescein diacetate (DCFDA), a fluorogenic dye, that measures hydroxyl, peroxyl and other ROS activity within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2′, 7′ –dichlorofluorescein (DCF). The cells were then analyzed using a Beckman Coulter CyAn ADP (Chapel Hill, USA).
Five hundred thousand cells were incubated with the vehicle, 1 μm ATO, 3 mM ASC, or 1 μm ATO plus 3 mM ASC. The cells were then harvested 48 h later and stained with the Annexin V-FITC apoptosis detection kit, according to manufactured instructions (eBioscience Ds, Bender Med Systems GmbH, Vienna, Austria), and analyzed using a Beckman Coulter CyAn ADP (Chapel Hill, USA). Intracellular glutathione (GSH) content was evaluated by a flow cytometry method based on the use of the thiol green dye, using the procedure recommended by the supplier (ABCAM, Co, USA).

Noguera N.I., Pelosi E., Angelini D.F., Piredda M.L., Guerrera G., Piras E., Battistini L., Massai L., Berardi A., Catalano G., Cicconi L., Castelli G., D’Angiò A., Pasquini L., Graziani G., Fioritoni G., Voso M.T., Mastrangelo D., Testa U, & Lo-Coco F. (2017). High-dose ascorbate and arsenic trioxide selectively kill acute myeloid leukemia and acute promyelocytic leukemia blasts in vitro. Oncotarget, 8(20), 32550-32565.

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Profiling Immune Cell Subsets by Flow Cytometry

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PBMCs were isolated using discontinuous Lymphoprep (Axis-ShieldPoCAs, Oslo, Norway) gradient centrifugation and were re-suspended in phosphate-buffered saline (PBS) containing 2% fetal bovine serum. Isolated PBMCs were collected by centrifugation, incubated with Fc-blocking antibody (purified anti-human CD16/32, BioLegend, San Diego, CA, USA) to prevent nonspecific binding and then incubated with the following antibodies (CD19: CF506236, BD Biosciences, CA, USA, 1:50; CD24: BDB563371, BD Biosciences, 1:50; CD27: BDB340424, BD Biosciences, 1:50; CD8: BDB564805, BD Biosciences, 1:50; IgA: ab193189, Abcam, CA, USA, 1:50) for 30 min on ice. After staining, cells were analyzed using CyAn-ADP (Beckman Coulter). Intracellular cytokine staining was performed by using the stimulation cocktail (eBioscience, San Diego, CA, USA) pre-stimulated for three hours as the manufacturer’s recommended protocol. Samples were analyzed by CyAn-ADP (Beckman Coulter) and data were processed using Flow Jo software (TreeStar, Ashland, OR).

Li W., Wang L., Shen C., Xu T., Chu Y, & Hu C. (2019). Radiation therapy-induced reactive oxygen species specifically eliminates CD19+IgA+ B cells in nasopharyngeal carcinoma. Cancer Management and Research, 11, 6299-6309.

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Measuring Cell Size and Proliferation

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For cell size measurement, cells were trypsinized, washed with ice-cold PBS, and their forward scatter values were measured by flow cytometry (CyAn ADP, Beckman Coulter, Brea, USA). The forward scatter (FSC-A) values of cells were analyzed by Modfit Software (Verity Software House, Turramurra, Australia).
Cell proliferation was analyzed by BrdU incorporation using anti-BrdU antibody, PE conjugated (339812, Biolegend, San Diego, USA). Briefly, cells at the logarithmic growth phase were cultured with 10 µM BrdU (B23151, Thermo Fisher) for 1 h, then harvested for ice-cold (-20 °C) 70% ethanol fixation for at least 2 hrs. For BrdU staining, cells were treated with 2 N HCl to denature DNA, and then neutralized with 0.1 mol/L Na₂B₄O₇. Then, the samples were incubated with anti-BrdU antibody (5ul per million cells) in PBS/5% BSA for 20 minutes at room temperature in the dark. After three washes in PBS, the nuclei were stained with DAPI (C1002, Beyotime, Haimen, China). Stained cells were analyzed by flow cytometry (CyAn ADP, Beckman Coulter).

Liu G., Wang Y., Zheng W., Cheng H, & Zhou R. (2019). P11 Loss-of-Function is Associated with Decreased Cell Proliferation and Neurobehavioral Disorders in Mice. International Journal of Biological Sciences, 15(7), 1383-1395.

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Characterizing EpCAM and Glucose Uptake

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Immunofluorescence staining was performed on plasma-membrane bound EpCAM on EBC1 cell line, RES-J EBC1 cells and CAF (2x10 5 ). Fluorescence intensity was measured by cytofluorimetric analysis (FACS analysis, CyAn ADP, Beckman Coulter s.r.l.) and analyzed using Summit Software 4.3. For the assay, cells were trypsinized, washed with PBS 2% FBS and stained with the EpCAM-FITC antibody (Miltenyi Biotec) for 20 minutes at room temperature. As negative control, cells were stained with a control IgG1-FITC antibody (Alexa Fluor).
For the glucose uptake assays cells were grown under normal conditions for 24 hr and 100 mM 2-NBDG (Invitrogen) was added to the media for 2 hours. The two gaussian tails (corresponding to the most and least glycolytic cells) were sorted (MoFlo ASTRIOS EQ di Beckman Coulter) and kept in culture. After one week cells were analysed again for glucose uptake through NBDG staining on FACS Cyan (CyAn ADP, Beckman Coulter s.r.l.) and analyzed using Summit Software 4.3.

Apicella M., Giannoni E., Fiore S., Ferrari K.J., Fernández-Pérez D., Isella C., Granchi C., Minutolo F., Sottile A., Comoglio P.M., Medico E., Pietrantonio F., Volante M., Pasini D., Chiarugi P., Giordano S, & Corso S. (2018). Increased Lactate Secretion by Cancer Cells Sustains Non-cell-autonomous Adaptive Resistance to MET and EGFR Targeted Therapies. Cell metabolism, 28(6).

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Flow Cytometric Analysis of Fixed Cells

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The cells were prepared for flow cytometry by growing them overnight at 30 °C. The cells were fixed using 95% ethanol, treated with 2 mg/mL RNAse (Sigma-Aldrich, St. Louis, MO, USA) for two hours at 37 °C and 5 mg/mL protease solution for 30 min at 37 °C, stained with 1 µM Sytox Green (ThermoFisher Scientific, Wilmington, DE, USA), and sonicated using a Diagenode Biorupter (Diagenode Corporation, Liège, Belgium) for 20 s on low power to disrupt clumps. Cells were analysed with 488 nm excitation on a Beckman Coulter CyAn^TM ADP flow cytometry machine (Beckman Coulter, CA, USA). Green fluorescence was collected at 523 nm. The data were analysed using Summit v4.3 software (Beckman Coulter, CA, USA).

Hewitt S.K., Duangrattanalert K., Burgis T., Zeef L.A., Naseeb S, & Delneri D. (2020). Plasticity of Mitochondrial DNA Inheritance and Its Impact on Nuclear Gene Transcription in Yeast Hybrids. Microorganisms, 8(4), 494.

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Cell Cycle Analysis by Flow Cytometry

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The cellular DNA content was analyzed from ethanol fixed cells (12,000 cells) by propidium iodide (PI) staining in a flow cytometer (CyAnTM ADP, Beckman Coulter, CA, USA). With this method, cells in the different phases of the cell cycle can be identified. Cells in subG1 phase, which indicates apoptotisis, can be identified as they contain fragmented DNA [64 (link)]. In the cell cycle analysis, the fixed cells were treated 0.15 mg ml⁻¹ of ribonuclease A and stained with PI (Sigma Aldrich Corp.) at a final concentration of 8 μg ml⁻¹. Etoposide (dose 2 μM) was used as a positive control in cell cycle analyses.

Muala A., Rankin G., Sehlstedt M., Unosson J., Bosson J.A., Behndig A., Pourazar J., Nyström R., Pettersson E., Bergvall C., Westerholm R., Jalava P.I., Happo M.S., Uski O., Hirvonen M.R., Kelly F.J., Mudway I.S., Blomberg A., Boman C, & Sandström T. (2015). Acute exposure to wood smoke from incomplete combustion - indications of cytotoxicity. Particle and Fibre Toxicology, 12, 33.

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Assaying PI Positive Cells Using Flow Cytometry

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The total amount of PI positive cells was assayed from freshly scraped cells using flow cytometry (CyAnTM ADP, Beckman Coulter, CA, USA). To separate cells and culture medium, the cell suspensions were centrifuged (370 × g, 5 min) and the cell pellet was resuspended in phosphate buffered saline (PBS). Cells were washed once with PBS before staining with PI (0.5 ml PBS, 1 μg/ml PI) Thereafter, cells were immediately analysed using excitation at 488 nm and emission filter 613 ± 20 nm (channel FL 3). A total of 12,000 cells were analysed for their PI content using Summit software version 4.3 (Beckman Coulter, CA, USA). PI positivity indicated compromised cell membrane.

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Cell Cycle Distribution and Apoptosis Analysis

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For cell cycle distribution and apoptosis analysis, cells were harvested and fixed in 70% ethanol at −20 °C overnight. They were then incubated with the staining buffer containing 36 μg/ml propidium iodide (Sigma) and 400 μg/ml RNase (Roche, Mannhein, Germany) at 37 °C for 20 min and analyzed by FCM (CyAnTM ADP, Beckman Coulter, Brea, CA, USA). Data were analyzed with ModFit LT software (Verity Software House, Topsham, ME, USA).

Zhang Q., Hou D., Luo Z., Chen P., Lv B., Wu L., Ma Y., Chu Y., Liu H., Liu F., Yu S., Zhang J., Yang D, & Liu J. (2015). The novel protective role of P27 in MLN4924-treated gastric cancer cells. Cell Death & Disease, 6(8), e1867-.

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Cell Cycle and Apoptosis Analysis

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Cells treated with MLN4924 or DMSO were harvested and fixed in 70% ethanol at -20°C overnight, and stained with propidium iodide (PI, 36 μg/ml, Sigma) containing RNAase (10 μg/ml, Sigma) at 37°C for 15 min, then analyzed for apoptosis and cell cycle profile by CyAnTM ADP (Beckman Coulter) as previously described [14 (link), 16 (link)]. Data were analyzed with ModFit LT software. Apoptosis was measured by the percentage of cells in sub-G1 population.

Gao Q., Yu G.Y., Shi J.Y., Li L.H., Zhang W.J., Wang Z.C., Yang L.X., Duan M., Zhao H., Wang X.Y., Zhou J., Qiu S.J., Jeong L.S., Jia L.J, & Fan J. (2014). Neddylation pathway is up-regulated in human intrahepatic cholangiocarcinoma and serves as a potential therapeutic target. Oncotarget, 5(17), 7820-7832.

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Myeloid Respiratory Burst Assay

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Differentiated PLB985 cells (5 × 10⁵) were stained with the myeloid marker CD11b-APC (clone M1/70, Biolegend). Cells were then incubated with 2.9 μM of DHR 123 (DHR; Sigma-Aldrich) in 500 μl of PBSgg (0.05% gelatin, 0.09% D-glucose) containing 150 U/ml of catalase (Sigma-Aldrich) for 15 min at 37 ^oC and subsequently activated with 1 μg/ml of PMA (Sigma-Aldrich) for further 15 min at 37 ^oC. Cells were kept on ice and analysed in CyAn^TM ADP (Beckman Coulter) within 30 min.
For murine cells, 50 μl of blood or 1 × 10⁶ bone marrow cells were stained with APC labelled anti-murine CD11b (clone M1/70, Biolegend) and anti-murine Gr-1 (clone RB6-8C5, Biolegend) antibodies before being subjected to a DHR test as above described. DHR positivity was calculated by gating on the cells with high SSC and high expression of CD11b/Gr-1 defined as granulocytes.

Schejtman A., Aragão-Filho W.C., Clare S., Zinicola M., Weisser M., Burns S.O., Booth C., Gaspar H.B., Thomas D.C., Condino-Neto A., Thrasher A.J, & Santilli G. (2020). Lentiviral gene therapy rescues p47phox chronic granulomatous disease and the ability to fight Salmonella infection in mice. Gene Therapy, 27(9), 459-469.

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