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Dako cyan flow cytometer

Manufactured by Agilent Technologies
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The Dako Cyan flow cytometer is a multiparameter, laser-based analysis instrument designed to identify and characterize cells based on their physical and fluorescent properties. It is capable of detecting and analyzing multiple fluorescent signals simultaneously, providing detailed information about the properties of individual cells within a sample.

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Lab products found in correlation

Live dead fixable near ir dead cell stain, by Thermo Fisher Scientific (3 mentions) Saponin, by Thermo Fisher Scientific (2 mentions) Il 17 pe, by Thermo Fisher Scientific (2 mentions) Cd4 fitc, by Thermo Fisher Scientific (2 mentions) Anti cd45ro fitc, by BD (2 mentions) Cd3 pe, by BD (2 mentions) Cd4 apc, by BD (2 mentions) Foxp3 staining buffer, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Fitc apoptosis detection kit 1, by BD (1 mentions)

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Cyan flow cytometer (38 citations) Cyan cytometer (6 citations) DAKO Flow Cytometer (2 citations)

10 protocols using dako cyan flow cytometer

1

Compound T Protects Renal Cells from Chemotherapy

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Example 169

Compound T Protects Renal Proximal Tubule Epithelial Cells from Chemotherapy-Induced DNA Damage

The ability of Cdk4/6 inhibitors to protect human renal proximal tubule cells from chemotherapy induced DNA damage was analyzed using etoposide and cisplatin. The cells were treated with Compound T in a dose dependent manner (10 nM, 30 nM, 100 nM, 300 nM, or 1000 nM). At the conclusion of the experiment, cells were harvested, fixed, and stained with propidium iodide (a DNA intercalator), which fluoresces strongly red (emission maximum 637 nm) when excited by 488 nm light. Samples were run on Dako Cyan flow cytometer. Data were analyzed using FlowJo 2.2 software developed by TreeStar, Inc. As seen in FIG. 21, results show that Compound T protects renal proximal tubule epithelial cells from chemotherapy induced DNA damage, as increasing dosages of Compound T in combination with etoposide or cisplatin cause a decrease in the percentage of S-phase cells, with a corresponding rise in the percentage of cells in the G0-G1 phase.

US09931345B2. Transient protection of normal cells during chemotherapy (2018-04-03). 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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2

Ex vivo Analysis of T-cell Phenotype

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CD45-RO + frequencies were assessed directly ex vivo by surface staining at 4 °C in PBS with antiCD45RO-FITC, CD3-PE and CD4-APC (all from BD Biosciences). For post-stimulation cultures, dead cells were labelled with near-IR LIVE/DEAD fixable dead cell stain (Molecular Probes, Life Technologies) before fixation. For analysis of regulatory markers: CTLA-4, Foxp3 and CD25, cells were fixed, permeabilised and stained with ebioscience/Thermofisher Foxp3 staining buffers according to the manufacturer's instructions. For analysis of cytokine expression, PMA/ionomycin-restimulated cells were fixed with 3% paraformaldehyde in PBS for 12 min followed by a 5-minute wash with PBS under centrifugation. Fixed cells were permeabilised with 0.1% saponin (Acros Organics) prepared in PBS and stained with IL-17-PE, IFNγ-e450, IL-21-APC, CD3-PERCP, CD4-FITC. For all studies cells were acquired on a Dako Cyan flow cytometer (Dako Cytomation) and data analysed using FlowJo software (Tree Star version 8.8.6). All antibodies were purchased from ebioscience/Thermofisher or BD Biosciences and expression quantified relative to the appropriate isotype control.
Jeffery L.E., Henley P., Marium N., Filer A., Sansom D.M., Hewison M, & Raza K. (2018). Decreased sensitivity to 1,25-dihydroxyvitamin D3 in T cells from the rheumatoid joint. Journal of Autoimmunity, 88, 50-60.
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3

Multiparametric Flow Cytometric Analysis of Immune Cells

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Dead cells were labelled with near-IR LIVE/DEAD fixable dead cell stain (Molecular Probes, Life Technologies) before fixation. For analysis of total CTLA-4, Foxp3 and CD25, cells were fixed, permeabilised and stained with ebioscience Foxp3 staining buffers according to the manufacturer’s instructions. For analysis of cytokine expression, cells were re-stimulated with PMA (50ng/ml), and ionomycin (1μM) for 5 hours, with Brefeldin A (10μg/ml) present during the last 4 hours (all from Sigma Aldrich). Cells were fixed with 3% paraformaldehyde in PBS for 12 minutes followed by a 5 minute wash with PBS under centrifugation. Cells were then permeabilised with 0.1% saponin (Acros Organics) prepared in PBS and stained with cytokine detection antibodies. Cells were acquired on a Dako Cyan flow cytometer (Dako Cytomation) and data analysed using FlowJo software (Tree Star). All antibodies were purchased from ebioscience or BD Biosciences and expression quantified relative to the appropriate isotype control.
Jeffery L.E., Qureshi O.S., Gardner D., Hou T.Z., Briggs Z., Soskic B., Baker J., Raza K, & Sansom D.M. (2015). Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines on CTLA-4 Expression and Regulatory Function. PLoS ONE, 10(7), e0131539.
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4

Cell Cycle Analysis of Renal Cells

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Example 168

DNA Cell Cycle Analysis in Human Renal Proximal Tubule Cells

To test the ability of Cdk4/6 inhibitors to induce a clean G1-arrest in non-hematopoietic cells, G1 arrest was examined in human renal proximal tubule cells. The cells were treated with Compound T in a dose dependent manner for 24 hours. At the conclusion of the experiment, cells were harvested, fixed, and stained with propidium iodide (a DNA intercalator), which fluoresces strongly red (emission maximum 637 nm) when excited by 488 nm light. Samples were run on Dako Cyan flow cytometer. Data were analyzed using FlowJo 2.2 software developed by TreeStar, Inc. Assays were run in triplicate, and error bars were not detectable. As seen in FIG. 20, results show that Compound T induces a robust G1 cell cycle arrest in human renal proximal tubule cells, as nearly all cells are found in the G0-G1 phase upon treatment with increasing amounts of Compound T.

US09931345B2. Transient protection of normal cells during chemotherapy (2018-04-03). 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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5

Cell Cycle Analysis of Human Renal Cells

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Example 168

DNA Cell Cycle Analysis in Human Renal Proximal Tubule Cells

To test the ability of Cdk4/6 inhibitors to induce a clean G1-arrest in non-hematopoietic cells, G1 arrest was examined in human renal proximal tubule cells. The cells were treated with Compound T in a dose dependent manner for 24 hours. At the conclusion of the experiment, cells were harvested, fixed, and stained with propidium iodide (a DNA intercalator), which fluoresces strongly red (emission maximum 637 nm) when excited by 488 nm light. Samples were run on Dako Cyan flow cytometer. Data were analyzed using FlowJo 2.2 software developed by TreeStar, Inc. Assays were run in triplicate, and error bars were not detectable. As seen in FIG. 20, results show that Compound T induces a robust G1 cell cycle arrest in human renal proximal tubule cells, as nearly all cells are found in the G0-G1 phase upon treatment with increasing amounts of Compound T.

US10660896B2. Transient protection of normal cells during chemotherapy (2020-05-26). G1 Therapeutics, Inc. [US]. Inventors: Jay Copeland Strum [US], John Emerson Bisi [US], Patrick Joseph Roberts [US], Francis X. Tavares [US].
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6

Compound T Protects Renal Cells from Chemotherapy Damage

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Example 169

Compound T Protects Renal Proximal Tubule Epithelial Cells from Chemotherapy-Induced DNA Damage

The ability of Cdk4/6 inhibitors to protect human renal proximal tubule cells from chemotherapy induced DNA damage was analyzed using etoposide and cisplatin. The cells were treated with Compound T in a dose dependent manner (10 nM, 30 nM, 100 nM, 300 nM, or 1000 nM). At the conclusion of the experiment, cells were harvested, fixed, and stained with propidium iodide (a DNA intercalator), which fluoresces strongly red (emission maximum 637 nm) when excited by 488 nm light. Samples were run on Dako Cyan flow cytometer. Data were analyzed using FlowJo 2.2 software developed by TreeStar, Inc. As seen in FIG. 21, results show that Compound T protects renal proximal tubule epithelial cells from chemotherapy induced DNA damage, as increasing dosages of Compound T in combination with etoposide or cisplatin cause a decrease in the percentage of S-phase cells, with a corresponding rise in the percentage of cells in the G0-G1 phase.

US10660896B2. Transient protection of normal cells during chemotherapy (2020-05-26). G1 Therapeutics, Inc. [US]. Inventors: Jay Copeland Strum [US], John Emerson Bisi [US], Patrick Joseph Roberts [US], Francis X. Tavares [US].
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7

Apoptosis and Cell Cycle Analysis

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Cells were plated onto 6-well culture plates, and then ZnO NP treatment was performed for 16 and 24 h. After each time point, cells were collected into a 15 mL centrifuge tube and pelleted by centrifugation at 1,000 rpm for 5 min. Cell pellets were washed twice with PBS, and subsequently cells were stained using Annexin V and fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (BD Biosciences). Briefly, cells were resuspended with 1× binding buffer at a concentration of 1×105 cells per 100 μL. Five microliters of FITC Annexin V and 5 μL propidium iodide (PI) were added and incubated for 15 min at room temperature (25°C) away from light. Samples were top up with another 400 μL of 1× binding buffer before analyzing by flow cytometry.
For cell cycle analysis, the cell pellet was fixed with ice cold ethanol overnight. The cells were washed and permeabilized with Triton-X 100, and then stained with PI. Samples were kept in the dark before being analyzed. A total of 1×104 events for each sample were analyzed and scored by Dako Cyan flow cytometer (DakoCytomation) supplemented with the Summit™ software.
Ng C.T., Yong L.Q., Hande M.P., Ong C.N., Yu L.E., Bay B.H, & Baeg G.H. (2017). Zinc oxide nanoparticles exhibit cytotoxicity and genotoxicity through oxidative stress responses in human lung fibroblasts and Drosophila melanogaster. International Journal of Nanomedicine, 12, 1621-1637.
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8

Quantifying Cell Surface Stress Proteins

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PBS-washed MCF-7cells were further washed with PBS containing 1 mM EDTA for additional 5 min. The cells were fixed in 4% paraformaldehyde (PFA) in PBS at 37° C for 30 min and then washed twice with PBS. Expression of cell surface proteins Hsc70, Hsp90, Hsp70, Hsp60, Hsp40, PDI, and integrin β3 was detected by incubating separately the cells with primary goat antibodies (0.2 µg/mL, Santa Cruz Biotechnology Inc.) to each cellular protein for 45 min at 4° C. Goat irrelevant antibody (IgG2a) was used as an isotype IgG control. After washing twice with PBS containing 1% BSA, the cells were incubated with donkey anti-goat IgG coupled to Alexa Fluor488 (0.4 µg/mL, Santa Cruz Biotechnology Inc. SC-362255) for 20 min at 4° C. After washing with PBS, the cells were analyzed using a Dako CyAn flow cytometer (Dako Cytomation). Data were presented as mean ( standard deviation from three independent experiments performed in duplicate.
Perez C., Rico J., Guerrero C, & Acosta O. (2021). Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates. Colombia Médica : CM, 52(1), e2024196.
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9

Flow Cytometry Characterization of MSCs

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For cell-surface marker characterization using flow-cytometry, 1 × 106 MSCs were stained with 1:100 dilution of fluorescein isothiocyanate-conjugated anti-CD34 (Santa Cruze, USA), CD45 (Chemicon; Hampshire, UK), CD90 (Abcam, Cambridge, UK) or Alexa 647-conjugated anti-CD29 (Biolegend, London, UK) antibodies. Corresponding isotype-matched control antibodies were used for negative controls. All antibodies were used at 1:100 dilution following instructions stipulated by the company's guidelines. Samples were analyzed using the Dako Cyan flow-cytometer (Dako Cytomation, UK).
Ichihara Y., Kaneko M., Yamahara K., Koulouroudias M., Sato N., Uppal R., Yamazaki K., Saito S, & Suzuki K. (2018). Self-assembling peptide hydrogel enables instant epicardial coating of the heart with mesenchymal stromal cells for the treatment of heart failure. Biomaterials, 154, 12-23.
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10

Phenotypic Analysis of T Cells

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CD45-RO+ frequencies were assessed directly ex vivo by surface staining at 4 o C in PBS with antiCD45RO-FITC, CD3-PE and CD4-APC (all from BD Biosciences). For post-stimulation cultures, dead cells were labelled with near-IR LIVE/DEAD fixable dead cell stain (Molecular Probes, Life Technologies) before fixation. For analysis of regulatory markers: CTLA-4, Foxp3 and CD25, cells were fixed, permeabilised and stained with ebioscience/Thermofisher Foxp3 staining buffers according to the manufacturer's instructions. For analysis of cytokine expression, PMA/ionomycinrestimulated cells were fixed with 3% paraformaldehyde in PBS for 12 minutes followed by a 5-minute wash with PBS under centrifugation. Fixed cells were permeabilised with 0.1% saponin (Acros Organics) prepared in PBS and stained with IL-17-PE, IFNγ-e450, IL-21-APC, CD3-PERCP, CD4-FITC. For all studies cells were acquired on a Dako Cyan flow cytometer (Dako Cytomation) and data analysed using FlowJo software (Tree Star version 8.8.6). All antibodies were purchased from ebioscience/Thermofisher or BD Biosciences and expression quantified relative to the appropriate isotype control.
Liu N.Q., Larner D.P., Yao Q., Chun R.F., Ouyang Y., Zhou R., Tamblyn J.A., Wagner C.L, & Hewison M. (2018). Vitamin D-deficiency and sex-specific dysregulation of placental inflammation. The Journal of steroid biochemistry and molecular biology, 177.
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