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

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.
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.
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Catalog Number22860
Unit Size
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)542
Emission (nm)580
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Excitation (nm)
542
Emission (nm)
580
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 (PE-Texas Red channel).

Platform


Flow cytometer

Excitation488 nm laser
Emission610/20 nm filter
Instrument specification(s)PE-Texas Red channel

Components


Component A: 500X Nuclear Red™ CCS21 Vial (100 µL)
Component B: Assay Buffer1 Bottle (50 mL)

Example protocol


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.

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.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)542
Emission (nm)580

References


View all 33 references: Citation Explorer
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, Hildrestr and 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
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
Videomicrofluorometry on living cells and discriminant factorial analysis to study cell cycle distributions
Authors: Savatier J, Gbankoto A, Vigo J, Salmon JM.
Journal: J Biol Regul Homeost Agents (2004): 206
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