Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*

Additional fluorescent color(s): 
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<p>DNA profile in growing Jurkat cells. Jurkat cells were stained with Nuclear Red™ CCS2 for 30 minutes. The fluorescence intensity of Nuclear Red™ CCS2 was measured using ACEA NovoCyte flow cytometer with the channel of PE-Texas Red, the image was generated using Cell Cycle Analysis module of NovoExpress software. In growing Jurkat cells, G0/G1 and G2/M phase histogram peaks are separated by a S-phase distribution.</p>
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Unit Size: Cat No: Price (USD): Qty:
100 tests 22860 $295


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)488/615
Storage F/D/L
InstrumentsFlow cytometer
Category Cell Analysis
Cell Cytotoxicity
Related Apoptosis and Cytotoxicity
Cell Apoptosis
Cell cycles have four sequential phases: G0/G1, S, G2, and M. During a cell's passage through cell cycle, its DNA is duplicated in S (synthesis) phase and distributed equally between two daughter cells in M (mitosis) phase. These two phases are separated by two gap phases: G0/G1 and G2. The two gap phases provide time for cells to grow and double the mass of their proteins and organelles. They are also used by cells to monitor internal and external conditions before proceeding with the next phase of cell cycle. The cell's passage through cell cycles is controlled by a host of different regulatory proteins. This assay kit is designed to monitor cell cycle progression and proliferation by using our proprietary Nuclear Red™ CCS2 in live cells. The percentage of cells in a given sample that are in G0/G1, S and G2/M phases, as well as the cells in the sub-G1 phase prior to apoptosis can be determined by flow cytometry. Cells stained with Nuclear Red™ CCS2 can be monitored with a flow cytometer at Ex/Em = 488 nm/615 nm (PE-Texas Red channel).




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells with test compounds at a density of 5 × 105 to 1× 106 cells/mL
  2. Add 1 µL of 500X Nuclear Red™ CCS2 into 0.5 mL of cell solution
  3. Incubate at 37 °C, 5% CO2 for 10 - 30 minutes
  4. Analyze cells using flow cytometer with PE-Texas Red channel (Ex/Em = 488 nm/615 nm)

Important notes
Thaw all the components at room temperature before starting the experiment.

Key parameters
Instrument:Flow cytometer
Excitation:488 nm
Emission:615 nm
Instrument specification(s):PE-Texas Red Channel

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Sample experimental protocol
  1. For each sample, prepare cells in 0.5 mL of warm medium or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density.

  2. Treat cells with test compounds for a desired period of time to induce apoptosis, cell cycle arrest or other cell cycle functions.

  3. Add 1 µL of 500X Nuclear Red™ CCS2 (Component A) in cells containing growth medium.

  4. Incubate the cells in a 37 °C, 5% CO2 incubator for 10 to 30 minutes. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Nuclear Red™ CCS2. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. It is not necessary to fix cells before staining since Nuclear Red™ CCS2 is cell- permeable.

  5. Optional: Centrifuge cells at 1000 rpm for 4 minutes and re-suspend cells in 0.5 mL of assay buffer (Component B) or a buffer of your choice.

  6. Monitor the fluorescence intensity using a flow cytometer with PE-Texas Red channel (Ex/Em = 488/615 nm). Gate the cells of interest, excluding debris.
Example data analysis and figures

Figure 1.

DNA profile in growing Jurkat cells. Jurkat cells were stained with Nuclear Red™ CCS2 for 30 minutes. The fluorescence intensity of Nuclear Red™ CCS2 was measured using ACEA NovoCyte flow cytometer with the channel of PE-Texas Red, the image was generated using Cell Cycle Analysis module of NovoExpress software. In growing Jurkat cells, G0/G1 and G2/M phase histogram peaks are separated by a S-phase distribution.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication
Authors: Ferullo DJ, Cooper DL, Moore HR, Lovett ST.
Journal: Methods (2009): 8

DNA replication, cell cycle progression and the targeted gene repair reaction
Authors: Engstrom JU, Kmiec EB.
Journal: Cell Cycle (2008): 1402

Morin inhibits the growth of human leukemia HL-60 cells via cell cycle arrest and induction of apoptosis through mitochondria dependent pathway
Authors: Kuo HM, Chang LS, Lin YL, Lu HF, Yang JS, Lee JH, Chung JG.
Journal: Anticancer Res (2007): 395

Direct control of cell cycle gene expression by proto-oncogene product ACTR, and its autoregulation underlies its transforming activity
Authors: Louie MC, Revenko AS, Zou JX, Yao J, Chen HW.
Journal: Mol Cell Biol (2006): 3810

Cell cycle markers for live cell analyses
Authors: Easwaran HP, Leonhardt H, Cardoso MC.
Journal: Cell Cycle (2005): 453

Dynamic relocalization of hOGG1 during the cell cycle is disrupted in cells harbouring the hOGG1-Cys326 polymorphic variant
Authors: Luna L, Rolseth V, Hildrestrand GA, Otterlei M, Dantzer F, Bjoras M, Seeberg E.
Journal: Nucleic Acids Res (2005): 1813

Dynamics of relative chromosome position during the cell cycle
Authors: Essers J, van Cappellen WA, Theil AF, van Drunen E, Jaspers NG, Hoeijmakers JH, Wyman C, Vermeulen W, Kanaar R.
Journal: Mol Biol Cell (2005): 769

Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis
Authors: Conlon KA, Zharkov DO, Berrios M.
Journal: DNA Repair (Amst) (2004): 1601

Description of a flow cytometry approach based on SYBR-14 staining for the assessment of DNA content, cell cycle analysis, and sorting of living normal and neoplastic cells
Authors: Nunez R, Garay N, Villafane C, Bruno A, Lindgren V.
Journal: Exp Mol Pathol (2004): 29

Differential roles of STAT1alpha and STAT1beta in fludarabine-induced cell cycle arrest and apoptosis in human B cells
Authors: Baran-Marszak F, Feuillard J, Najjar I, Le Clorennec C, Bechet JM, Dusanter-Fourt I, Bornkamm GW, Raphael M, Fagard R.
Journal: Blood (2004): 2475


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Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Cell Apoptosis & Proliferation

Certificate of Analysis