Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit Optimized for 405 nm Violet Laser Excitation

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Spectral properties

Excitation (nm)	401
Emission (nm)	460

Storage, safety and handling

Intended use

Research Use Only (RUO)

Alternative formats

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

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

Overview SDS Protocol

Excitation (nm)

401

Emission (nm)

460

The cell cycle has 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 the cell to grow and double the mass of their proteins and organelles. They are also used by the cells to monitor internal and external conditions before proceeding with the next phase of cell cycle. The cell's passage through cell cycle is controlled by a host of different regulatory proteins. This particular kit is designed to monitor cell cycle progression and proliferation by using our proprietary Nuclear Violet™ 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 Violet™ can be monitored with a flow cytometer (Pacific Blue® channel).

Platform

Flow cytometer

Excitation	405 nm laser
Emission	450/40 nm filter
Instrument specification(s)	Pacific Blue channel

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
Add 1 µL of 500X Nuclear Violet™ into 0.5 mL of cell solution
Incubate at room temperature for 30 - 60 minutes
Analyze with a flow cytometer using the 405 nm violet laser excitation

Important notes
Thaw all the components at room temperature before use.

SAMPLE EXPERIMENTAL PROTOCOL

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 for apoptosis induction.
Treat cells with test compounds for a desired period of time to induce apoptosis or other cell cycle functions.
Add 1 µL of 500X Nuclear Violet™ (Component A) into the cells containing the growth medium, and incubate the cells in a 37°C, 5% CO₂ incubator for 30 to 60 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 Violet™. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. Note: It is not necessary to fix the cells before DNA staining since the Nuclear Violet™ is cell-permeable. Note: Human and rodent stem cells efflux Nuclear Violet™ stain, and this is the basis for the Side Population (SP) technique; the efflux can be blocked with blocking agents like verapamil, fumitermorgin C to prevent dye efflux for accurate DNA content analysis.
Optional: Centrifuge the cells at 1000 rpm for 4 minutes, and then re-suspend cells in 0.5 mL of assay buffer (Component B) or the buffer of your choice.
Monitor the fluorescence intensity by flow cytometry using the 405 nm violet laser (Ex/Em = 405/445 nm). Gate on the cells of interest, excluding debris.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	401
Emission (nm)	460

Product Family

Name	Excitation (nm)	Emission (nm)
Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit Red Fluorescence Optimized for Flow Cytometry	537	618

Images

Figure 1. DNA profile in growing Jurkat cells with Cell Meter™ Fluorimetric Cell Cycle Assay Kit. Jurkat cells were stained with Nuclear Violet™ for 30 minutes. The fluorescence intensity of Nuclear Violet™ was measured using ACEA NovoCyte flow cytometer with the channel of Pacific Blue. In growing Jurkat cells, G0/G1 and G2/M phase histogram peaks are separated by the S-phase distribution.

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