Navigation Links
Quantitative Analysis of the Cell Cycle in Imaging Applications

Introduction

Quantitative analysis of stages in the cell cycle is commonly used in cell biology since many pharmacological interventions aim to modulate the cell cycle in some way. We present here an application that can be used in adherent cells and optionally multiplexed for the use in automated cell signaling analysis and drug-screening applications.

The cell cycle consists of a series of processes involved in the growth, replication, and division of cells and can be divided into two major stages, interphase (the phase between mitotic events) and mitosis. There are three distinct, successive stages within interphase, namely G1, S, and G2 phases. During G1 (first gap), cells monitor their environment and grow (synthesize RNA and proteins). Once conditions are optimal, cells commit to DNA synthesis (S phase) and replicate their chromosomal DNA. The G2 phase (second gap) follows when cells continue to grow and prepare for mitosis (M phase, division). The G2 gap allows time for the cell to complete its DNA replication before initiating mitosis.

During mitosis, the cell cycle is completed giving rise to two daughter cells, each of which contains the same genetic material as the original cell and approximately half of its G2 level of cytoplasm. In addition to these specific stages, a G0 phase has been described for cells that exit from the cellcycle and enter a quiescent, non-dividing state. In response to external stimuli, cells may undergo reactivation and leave G0 and enter the G1 phase of the cell-cycle. Another consequence of cellular activation may be the induction of programmed cell death (apoptosis).

Understanding the specific phase of a cell is important when cells display heterogeneous responses to drugs as is often the case in high-content applications.

This four color application demonstrates how fluorescent antibodies can be used in combination to specifically inform on the M and S phases of the cell cycle while it is still possible to drop in an additional specificity of interest to the user. For this assay, Hoechst 33342 was used to identify all nuclei and an anti-phospho-Histone 3 antibody was used to identify cells in M phase.1 Similarly immunofluorescent staining of incorporated bromodeoxyuridine (BrdU) is used to determine the frequency of cells that had synthesized DNA.2, 3, 4, 5 In this method, BrdU (an analog of the DNA precursor thymidine) is incorporated into newly synthesized DNA by cells entering into and progressing through the S phase of the cell cycle. The drop in choice for this assay was a fluorescently labeled anti-Ki-67 antibody. Ki-67 is present in all cycling cells but is not present in quiescent cells (G0). The Ki-67 staining pattern is variable depending on the phase of the cycle, in the G1/S/G2 phases it is punctuate, whereas in late G2/M it appears throughout the entire nucleus (see Table 1 ).6,7

The BD Pathway Bioimager is well suited for the development of complex assays due to its wide range of acquisition and analysis tools. Using a broad-spectrum illumination source, the system enables the user to select the antibodies based on their compatibility with the assay rather than with specific laser wavelengths. The 16 excitation filter choices of the BD Pathway Bioimager allow the setup of several multi-color assays with minimal bleed-through which enables the system to be used in a wide variety of demanding applications.

Methods

HeLa cells (a human cervical cancer cell line, ATCC, #CCL-2) were seeded in 96-well clear bottom tissue culture plates optimized for automated imaging applications (Cat. No. 35-3219) at 15,000 cells/well in DMEM (Invitrogen, 11965-084) complete media. Eighteen hours later cells were treated with one of two different metabolic inhibitors, aphidicolin (Sigma-Aldrich, A0781), a DNA topoisomerase alpha inhibitor (G1/S blocker) for two hours or colcemid (Sigma-Aldrich Cat # D7385) a (G2/ M blocker for two hours. During drug treatment, cells were cocultured with 20 μM BrdU (Calbiochem, 203806) (a thymidine analogue that incorporates into DNA) for the final hour of culture.

For image analysis, cells were fixed and permeabilized in BD CytoFix/CytoPerm (4% paraformaldehyde with 0.1% saponin, Cat. No. 552598a) for 20min, washed with BD Perm/Wash Buffer (PBS with 0.1% saponin and 3% fetal calf serum, FCS, Cat. No. 552598b), and permeabilized again in BD CytoPerm Plus Permeabilization Buffer (BD 552598c) for 10 min. Afterwards, they were washed with the Perm/Wash Buffer, and re-fixed for 5 minutes. Cells were then washed with Perm/Wash Buffer, treated with DNAse (Sigma-Aldrich, D4513, 15μg/well in PBS at 37C for 1 hour), washed in Perm/Wash Buffer and stained simultaneously with Alexa Fluor 488 (Invitrogen, A-20000) conjugated anti-BrdU (Cat. No. 555627). Alexa Fluor 647 conjugated anti phospho-Histone H3 (Serine 28) (Cat. No. 558217), and Alexa Fluor 555 (Invitrogen A- 20009) conjugated anti-Ki-67 (Cat. No. 610969) in PBS with 3% FCS at room temperature for 1 hour. Cells were washed three times in Perm/Wash Buffer and stained with 2 μg/mL Hoechst 33342 (Invitrogen, H3570), in Perm/Wash Buffer

Images were acquired on a BD Pathway Bioimager and analyzed using Attovision software and BD Image Data Explorer.

Cells were imaged using a 20 (0.75 NA) objective and analyzed using the built-in tools of the software package. Numerical data was imported into the Microsoft Excelbased BD Image Data Explorer where the cell population was analyzed for nuclear fluorescence intensity by setting a threshold for the three fluorescence channels. The threshold was set on the nuclear intensity of the BrdU and phospho-Histone 3 negative cells.

Results

The staining pattern for BrdU and phospho-Histone H3 found in control cells was consistent with a heterogeneous cell population ( Figure 1 ). We found that approximately 3% were in M phase based on the positive phospho-Histone 3 staining and approximately 22% were in S phase based on the BrdU results ( Figure 1D ). When cells were treated with either colcemid or aphidicolin changes in cell cycle status were seen ( Figure 1 and Figure 3 ). In colcemid treated cells (eg, 500 ng/mL) the percent cells in S phase remained similar (~20%) while the percent cells in M phase doubled to approximately 7% ( Figure 1D ). In aphidicolin treated cells (eg, 500ng/mL), the percent cells in S phase dropped to 0% while the percent cells in M phase dropped slightly to approximately 2%. Figure 2 shows the dose-response relationship of BrdU incorporation in response to increasing concentrations of colcemid and aphidicolin. BrdU indicates cells in S phase and colcemid had no significant effect at any concentration. Aphidicolin decreased the number of S-phase cells in a dose-dependent manner.

Discussion

To assure homogenous responses in cellular assays, cells are often synchronized using multiple rounds of cell-cycle arrest and release. This adds complexity and unreliability to the assay in question and is incompatible with many assays especially stem cell or primary cell assays. We therefore developed a versatile application that utilizes standard antibody treatment to identify the cell cycle phases and applied it to automated imaging. As a stand-alone application, the assay quantifies the degree to which cells are arrested in the different cycle phases after drug treatment making it useful in oncology and other fields where drug-responses are suspected to be dependent on the specific cell cycle phase. In addition, the nature of the exchangeable antibodies allows this assay to be multiplexed with other applications. For example, the Ki-67 stain could be substituted with a marker for pathway activation. Therefore this assay can work as a system of building blocks where users can choose their preferred dyes and antibodies in combination to generate higher dimensional data sets.

References

1. Endl, E., P. Steinbach, R. Knu chel and F. Hofstadter. 1997. Analysis of cell cycle-related Ki-67 and p120 expression by flow cytometric BrdUrd-Hoechst/7AAD and immunolabeling technique. Cytometry . 29:233-241.

2. Goto, H., Tomono Y., Ajiro, K., et al.. 1999. Identification of a novel phosphorylation site on the histone H3 coupled with mitotic chromosome condensation . J. Biol. Chem 274, 25543-25549.

3. Kill, I.R. 1996. Localization of the Ki-67 antigen within the nucleolus. J. Cell Science. 109:1253-1263.

4. Miltenburger, H. G., G. Sachse and M. Schliermann. 1987. S-phase cell detection with a monoclonal antibody. Dev. Biol. Stand. 66:91-99.

5. Moran, R., Z. Darzynkiewicz, L. Staiano-Coico and M. R. Melamed. 1985. Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step. J. Histochem. Cytochem. 33:821-827.

6. Shapiro, H. M., Practical Flow Cytometry, 3rd Edition, Wiley-Liss, New York. Vanderlaan, M. and C. B. Thomas. 1985. Characterization of monoclonal antibodies to bromodeoxyuridine. Cytometry. 6:501-505.

7. Starborg, M., K. Gell, E. Brundell, and C. Hg. 1996. The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression. J. Cell Science. 109:145-153.


'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Mx4000 Multiplex Quantitative PCR System*,PPP
2. Improved Quantitative Selectivity of Clenbuterol in Human Urine using High Resolution on the TSQ Quantum Mass Spectrometer
3. The LightCycler is the only System for Quantitative PCR that is as Flexible as You are!
4. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
5. Quantitative end-point RT-PCR gene expression measurement using the Agilent 2100 Bioanalyzer and standardized RT-PCR
6. Detection of Genetically Modified Soybean in Processed Foods Using Real-Time Quantitative PCR with SYBR Green I Dye on the DNA Engine Opticon 2 System
7. Qualitative and Quantitative Assessment of Plasmid DNA Isolation, Rev A
8. Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A
9. Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ Real-Time PCR Detection System and iQ Supermix, Rev A
10. Quantitative Analysis of GFP in Plant Extracts Using the VersaFluor Fluorometer System
11. Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Quantitative Analysis the Cell Cycle Imaging Applications

(Date:12/24/2014)... Nashville Fertility Center announced that Dr. ... American Society for Reproductive Medicine (ASRM), a leading professional ... medicine. In his new role as treasurer, Dr. ... and will have a voice in furthering the mission ... ASRM since 1984 when he joined while conducting fellowship ...
(Date:12/24/2014)... December 23, 2014 Pipette.com announces their ... Twitter followers can submit their #HolidayInTheLab pictures for a ... lab mates. , For those struggling to think of ... Pipette.com Holiday in the Lab Contest provides the ideal ... followers to send pictures of their chemistree, holiday ...
(Date:12/24/2014)... 23, 2014 Vermillion, Inc. (Nasdaq:   ... disease, announced today that investors including Oracle Investment ... LLC and several Vermillion directors have agreed to ... Vermillion,s common stock and warrants to purchase unregistered ... placement.  Under the terms of ...
(Date:12/24/2014)... Dec. 23, 2014 Sigma-Aldrich Corporation (NASDAQ: ... waiting period under the Hart-Scott-Rodino Antitrust Improvements Act ... on December 22, 2014, thereby completing the U.S. ... the acquisition of the Company by Merck KGaA, ... satisfies another condition to closing the transaction, which ...
Breaking Biology Technology:Nashville Fertility Center Announces Dr. George A. Hill Selected to Serve as ASRM Treasurer for 2014-15 2Nashville Fertility Center Announces Dr. George A. Hill Selected to Serve as ASRM Treasurer for 2014-15 3Major Pipette Distributor Pipette.com Announces their Holiday in the Lab Contest 2Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 2Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 3Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 4Vermillion Announces Equity Financing of up to $18.9 Million; Suspends ATM Program 5Acquisition of Sigma-Aldrich by Merck KGaA, Darmstadt, Germany, Receives U.S. Antitrust Clearance 2Acquisition of Sigma-Aldrich by Merck KGaA, Darmstadt, Germany, Receives U.S. Antitrust Clearance 3
... (Nasdaq and SWX: BMRN) announced today the appointment,of ... Former Vice President of,Research and Development for Raven ... patents and the recipient of a,collection of prestigious ... Awards from Intellectual Property Owners, Inc., "We ...
... Therapeutics Inc., an,early stage biotechnology company developing therapies ... Drug application (IND),to the U.S. Food and Drug ... the first-in-man clinical trials for its lead compound,VEL-0230., ... has dual-acting properties, in,that it both stimulates bone ...
... N.J., Jan. 28 Ferring Pharmaceuticals Inc.,has announced ... will serve as the title sponsor of the ... GeezerJock of the Year,Award., "This is such ... for,senior athletes," said Alex Drigan, Product Manager, EUFLEXXA(TM). ...
Cached Biology Technology:BioMarin Appoints Dr. Gordon Vehar, Vice President of Research 2BioMarin Appoints Dr. Gordon Vehar, Vice President of Research 3Velcura Therapeutics, Inc. Can Begin Clinical Trials Following Successful Investigational New Drug Application to U.S. FDA 2Ferring Pharmaceuticals Announces EUFLEXXA(TM) as Title Sponsor of the National Senior Games Hockey Championships and the GeezerJock of the Year Award 2Ferring Pharmaceuticals Announces EUFLEXXA(TM) as Title Sponsor of the National Senior Games Hockey Championships and the GeezerJock of the Year Award 3Ferring Pharmaceuticals Announces EUFLEXXA(TM) as Title Sponsor of the National Senior Games Hockey Championships and the GeezerJock of the Year Award 4
(Date:12/19/2014)... Dec. 18, 2014   LaunchKey , the first ... post-password and Internet of Things era, today announced the ... venture round was led by Metamorphic Ventures with participation ... Partners, VegasTechFund, and others.  LaunchKey has raised $4 million ... expand its team and bring LaunchKey to market in ...
(Date:12/17/2014)... YORK , Dec. 15, 2014  HITLAB ... Good Clinical Practices (GCP) audit to confirm its ... (FDA) regulations. This accomplishment enables HITLAB to conduct ... utilizing the highest principles for patient safety and ... improve global healthcare access, quality, and delivery with ...
(Date:12/17/2014)... 2014  Automation is fundamentally transforming the travel ... at international borders. Over the past decade, ePassports, ... veteran travelers to self process through border control ... at an increasing number of airports, seaports, and ... According to Maxine Most , Principal at ...
Breaking Biology News(10 mins):LaunchKey Raises $3 Million in Additional Funding Led by Metamorphic Ventures 2LaunchKey Raises $3 Million in Additional Funding Led by Metamorphic Ventures 3HITLAB Completes GCP Audit for FDA Smart Device & App Compliance 2Automated Border Control (ABC) Transforms the Global Travel Experience With More Than 2500 ABC eGates and APC Kiosks Deployed In Airports, Seaports, and Land Borders Worldwide 2
... Rehabilitation Medicine at NYU Langone Medical Center will ... rehabilitation, the treatment of traumatic brain injuries and ... at the American Association of Physical Medicine and ... 2011. Experts will be available to comment about ...
... (University of Turku and VTT Technical Research Centre of Finland) ... Institute for Health and Welfare) have discovered that the SHARPIN ... leading journal, the study concludes that SHARPIN regulates the movement ... cancer cells. It is likely that the discovery will also ...
... Palo Alto, CAU.S. scientists have developed a new, integrated, ... carbon cycle and people,s role in it. Understanding the carbon ... sustainable future. The plan builds on the first such plan, ... the role of humans as agents and managers of carbon ...
Cached Biology News:Rusk experts present at American Association of Physical Medicine and Rehabilitation meeting 2Scientists tackle the carbon conundrum 2
... low melting point agarose used for second-dimension ... in place for most applications. Bromophenol Blue ... of electrophoresis runs. The solution is composed ... 1x Tris, glycine, SDS, and 0.003% Bromophenol ...
The Trans-Blot Plus gel and cassette assembly tray is used to hold the Trans-Blot Plus gel holder cassette, buffer, and other materials for the gel and membrane sandwich during their assembly....
... IEF cell is an apparatus dedicated to ... or agarose slab gels. The cell includes ... casting tray, 5 glass plates, 50 sheets ... sample templates, and instructions. Dimensions are 21 ...
... The Biomek FXP is the latest entry ... for greater positional accuracy and increased robustness, it ... application. It can be configured with either one ... capacity, the Biomek FXP sets the standard for ...
Biology Products: