Method For Cell Cycle Analysis Research Articles

Nanocomposites of iron oxide, sodium alginate, and eugenol induce apoptosis via PI3K/Akt/mTOR signaling in Hep3 cells and in vivo hepatotoxicity in the zebrafish model

Hepatic cancer is among the most recurrently detected malignancies worldwide and one of the main contributors to cancer-associated mortality. With few available therapeutic choices, there is an instant necessity to explore suitable options. In this aspect, Nanotechnology has been employed to explore prospective chemotherapeutic approaches, especially for cancer treatment. Nanotechnology is concerned with the biological and physical properties of nanoparticles in the therapeutic use of drugs. In the current work, formulation, and characterization of α-Fe2O3–Sodium Alginate-Eugenol nanocomposites (FSE NCs) using several approaches like SEM and TEM, UV–visible, FTIR, and PL spectroscopy, XRD, EDAX, and DLS studies have been performed. With an average size of 50 nm, the rhombohedral structure of NCs was identified. Further, their anticancer activity against Hep3B liver cancer cell lines has been performed by cell viability, dual staining, DCFH-DA, Annexin-V/-FITC/PI, cell cycle analysis methods, and PI3K/Akt/mTOR signaling proteins were studied to assess the anticancer effects of the NCs in Hep3B cells. Also, anti-cancer activity on animal modeling in-vivo using zebra fishes to hematological parameters, liver enzymes, and histopathology study effectiveness was noticed. Moreover, the NCs reduced the viability, elevated the ROS accumulation, diminished the membrane integrity, reduced the antioxidants, blocked the cell cycle, and triggered the PI3K/Akt/mTOR signaling axis that eventually resulted in cell death. As a result, FSE NCs possess huge potential for use as a possible anticancer candidate.

The role of miR-199a-3p in inhibiting the proliferation of spermatogonial stem cells under heat stress

MicroRNAs (miRNAs) play a crucial role in regulating various physiological processes, including cell differentiation, proliferation, and apoptosis. However, their specific functions in response to heat stress are not fully understood. This study aimed to investigate the regulatory effects of miR-199a-3p on the proliferation of heat stress-treated spermatogonial stem cells (SSCs). SSCs were isolated from mouse testes and cultured in vitro to identify marker molecules. Lentiviruses carrying miR-199a-3p-over, miR-199a-3p-inhibit, and ID4-over constructs were generated for stable transfection. Luciferase assay was employed to confirm the targeting relationship between miR-199a-3p and ID4. An in vitro SSCs heat stress model was established, and the miR-199a-3p-inhibit and ID4-over groups were included. Cellular proliferation was assessed using CCK-8, EdU, and cell cycle analysis methods after heat stress. Expression levels of miR-199a-3p and ID4 were evaluated by western blotting and qRT-PCR. The results demonstrated that miR-199a-3p-over inhibited SSCs proliferation, while ID4-over promoted an increase in SSCs number. Luciferase assay confirmed the regulatory effect of miR-199a-3p on ID4 expression. Moreover, after heat stress treatment, miR-199a-3p-inhibit and ID4-over enhanced SSCs proliferation compared to the control group. These findings suggest that miR-199a-3p modulates SSCs proliferation by targeting ID4, especially under heat stress conditions.

Basic Methods of Cell Cycle Analysis.

Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.

New Methods for Cell Cycle Analysis

Abstract Cell cycle is a basic process during the cell life. Analyzing cell cycles can better understand the nature of diseases. Currently, cell cycles are mainly analyzed by flow cytometry to investigate intracellular DNA contents and cell cycle specific proteins. With the development of technology, several new methods have emerged for cell cycle analysis in recent years to improve and enhance image analysis, single-cell analysis, sample reduction, and high throughput. These new methods include image flow cytometry, mass flow cytometry, microfluidic technology, and Raman spectroscopy, which have greatly expanded the methods for cell cycle analysis and increased the accuracy of cell cycle measurements. Here we briefly introduce these new methods for cell cycle analysis.

Microfluidic Cell Cycle Analysis of Spread Cells by DAPI Staining

Single-cell cell cycle analysis is an emerging technique that requires detailed exploration of the image analysis process. In this study, we established a microfluidic single-cell cell cycle analysis method that can analyze cells in small numbers and in situ on a microfluidic chip. In addition, factors that influenced the analysis were carefully investigated. U87 or HeLa cells were seeded and attached to microfluidic channels before measurement. Cell nucleic DNA was imaged by 4′-6-diamidino-2-phenylindole (DAPI) staining under a fluorescent microscope and subsequently fluorescent intensities of the cell nuclei DNA were converted to depict histograms for cell cycle phases. DAPI concentration, microscopic magnification, exposure time and cell number were examined for optimal cell cycle analysis conditions. The results showed that as few as a few hundred cells could be measured by DAPI staining in the range of 0.4–0.6 μg/mL to depict histograms with typical cell cycle phase distribution. Microscopic magnification during image acquisition, however, could distort the phase distribution. Exposure time did not significantly affect the cell cycle analysis. Furthermore, cell cycle inhibitor rapamycin treatment changed the cell cycle phase distribution as expected. In conclusion, a method for microfluidic single-cell cell cycle analysis of spread cells in situ was developed. Factors such as dye concentration and microscopic magnification had more influence on cell cycle phase distribution. Further studies will focus on detail differentiation of cell cycle phases and the application of such a method for biological meanings.

Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers.

The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in mitosis, G1, S and G2 stage in a single assay. The protocol detailed here employs detergent-based hypotonic solution to rapidly permeabilize cells and allows simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3, with specific antibody within 20 min. The protocol requires only inexpensive and commercial available reagents and also enables a rapid and complete analysis of cell cycle profile.

Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point “snapshot” of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes—cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)—as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods.

High throughput cell cycle analysis using microfluidic image cytometry (μFIC)

Microfluidic image cytometry (μFIC) is a novel approach for the cytotoxicity assessment of the cells cultured and treated within microfluidic channels under a precisely controlled chemical environment. Here, following our previous morphology-, and MTT absorbance-based μFIC, we are presenting our recent effort to develop, evaluate, and apply a high throughput cell cycle analysis method using fluorescence-based μFIC. A microfluidic device with a concentrantion gradient generator (CGG) and eight straight cell culture channels was fabricated, optimized, and applied for the assessment of paclitaxel-induced cell cycle changes of HeLa cells. Throughout this study, we have shown that the cell cycle analysis using fluorescence-based μFIC was able to provide comparable experimental data with those of flow cytometry. Moreover, cell cycle analysis using μFIC can also provide further advantages over flow cytometry, such as higher throughput, lower assay cost, less generation of toxic waste, and etc., which should have significant implications in pharmaceutical and biological applications as a future high throughput cell cycle analysis platform.

A robust flow‐cytometric protocol for assessing growth rate of hatchery‐reared barramundiLates calcariferlarvae

In this study, a flow-cytometric cell cycle analysis method to assess instantaneous growth rate of whole larvae of the Australian barramundi Lates calcarifer was developed and validated. High-resolution DNA measurements of either fresh, frozen or RNAlater-preserved larvae (gap0-gap1, G(0) -G(1), coefficient of variation (c.v.) < 3, 4 and 5%, respectively) enabled the deconvolution of the DNA histogram and assignment of the proportion of nuclei into cell cycle compartments G(0) -G(1), S (DNA synthesis) and G(2) -M (Gap2-Mitosis). This technique can be also used for individual fish tissues such as brain, liver, fin and muscle. For the first time, the combined proportion of replicating nuclei (into S and G(2) -M phases) of whole fish larvae and absolute growth rate in length (mm day(-1)) has been correlated in commercial aquaculture conditions. Fast growing L. calcarifer larvae had an overall hyperplasia advantage as indicated by a greater proportion of cells in the S+G(2) -M phase compared with slow growing larvae, which might explain the increasing differences in size during culture. In a fasting trial, larvae ceased growth while maintaining the constant initial rates of cell division throughout a 6 day period. For a highly fed fast growing control group, cell division rates significantly increased after day 4. Flow-cytometric cell cycle analysis of whole fish larvae may provide fish biologists and aquaculturists with a better understanding of how cell division rates influence early growth in natural and artificial environments.

Abstract 2962: A rapid alternative method for cell cycle analysis using the Cellometer Vision

Abstract Traditional cell cycle analysis instrumentation involves the use of fluorescence microscopy, laser scanning cytometry, and flow cytometry. In this work, we demonstrate a rapid alternative device, the Cellometer Vision, which utilizes an imaging cytometry method for analyzing cell cycle measurement. This method employs both bright-field and fluorescence imaging of a disposable cell counting chamber to quickly provide concentration and percentages of cell subpopulations. Signal measurement and data analysis could be performed less than five min. Cell cycle assay was demonstrated by detection of changes in phase percentage within a cell population when Jurkat cells were induced with three different drugs, nocodazole, etoposide, and aphidicolin that arrest cell cycle at G2/M, S, and G0/G1 phase, respectively. Jurkat cells were fixed with 75% ethanol and stained with propidium iodide. Individual cells were identified by the software and the fluorescence intensity was measured from within the cell. The imaging cytometry method was able to detect and analyze fluorescence from the Jurkat cells, which generated cell cycle data plot for each drug at three different concentrations. The cell cycle analysis results were highly consistent with the results obtained from FACScalibur flow cytometer. This imaging cytometry method provides a rapid and cost-effective tool for cell concentration measurements, as well as cell cycle analysis, which may benefit smaller research laboratories, where flow cytometer is not readily available. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2962. doi:10.1158/1538-7445.AM2011-2962

The Combination of 17-Estradiol and Bisphenol A Weaken their Respective Action

17-Estradiol (E2) and Bisphenol A (BPA) are important endocrine disrupting chemicals (EDCs). In this work, cell proliferation assay using cell cycle analysis method and determination of estrogen receptor α protein was used to investigated the mechanism and effects respectively and in mixture of the two EDCs in stimulating the growth of human breast cancer MCF-7 cell When the concentration of E2 and BPA were 10-12mol/L and 10-8mol/L respectively, E2 and BPA could reduce the express of ERα protein by activated ERα pathway, accelerated the cell cycle, decreased the percentages of cells in G0/1 phase, increased the percentages of cells in S phase at 48 h, and thus promoted the cancer cell growth. However, the combination effect of these two compounds did not induce the growth of cancer cell and the mixture reduced the ratio in S phase compared with E2 or BPA alone, which lead to weakening of the proliferation ability. There was a significant interaction between BPA and E2 and such a weakening effect had not been reported before.

A signature-based method for indexing cell cycle phase distribution from microarray profiles

BackgroundThe cell cycle machinery interprets oncogenic signals and reflects the biology of cancers. To date, various methods for cell cycle phase estimation such as mitotic index, S phase fraction, and immunohistochemistry have provided valuable information on cancers (e.g. proliferation rate). However, those methods rely on one or few measurements and the scope of the information is limited. There is a need for more systematic cell cycle analysis methods.ResultsWe developed a signature-based method for indexing cell cycle phase distribution from microarray profiles under consideration of cycling and non-cycling cells. A cell cycle signature masterset, composed of genes which express preferentially in cycling cells and in a cell cycle-regulated manner, was created to index the proportion of cycling cells in the sample. Cell cycle signature subsets, composed of genes whose expressions peak at specific stages of the cell cycle, were also created to index the proportion of cells in the corresponding stages. The method was validated using cell cycle datasets and quiescence-induced cell datasets. Analyses of a mouse tumor model dataset and human breast cancer datasets revealed variations in the proportion of cycling cells. When the influence of non-cycling cells was taken into account, "buried" cell cycle phase distributions were depicted that were oncogenic-event specific in the mouse tumor model dataset and were associated with patients' prognosis in the human breast cancer datasets.ConclusionThe signature-based cell cycle analysis method presented in this report, would potentially be of value for cancer characterization and diagnostics.

A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle

Objective: To set up a three-parameter method for cell cycle analysis by two-laser flow cytometer, which can detect two types of cyclin plus DNA content in one measurement, and that analyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysis of two types of cyclins and DNA content simultaneously in individual cells by two-laser flow cytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. A Triton-X100 permeabilization procedure was optimized for detection of two types of cyclins. One cyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other, indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochrome DAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method. Results: Permeabilization with 0.5% Triton-X100 in PBS containing 1% BSA for 5 min on ice provided optimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It was found that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured with no compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growing MOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously, allowing for analysis of cell cycle phase specific perturbations without the necessity of cell synchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine and cyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleled expected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only be applied to measure unscheduled vs. expected cyclin expression but may also be used to estimate the fraction of cycling cells in up to 6 cell populations.

CSX/Nkx2.5 Modulates Differentiation of Skeletal Myoblasts and Promotes Differentiation into Neuronal Cells in Vitro

CSX/Nkx2.5 transcription factor plays a pivotal role in cardiac development; however, its role in development and differentiation of other organs has not been investigated. In this study, we used C2C12 myoblasts and human fetal primary myoblasts to investigate the function of Nkx2.5 in skeletal myogenesis. The expression levels of Nkx2.5 decreased as C2C12 myoblasts elongated and fused to form myotubes. The expression of human NKX2.5 in C2C12 myoblasts inhibited myocyte differentiation and myotube formation, and up-regulated Gata4 and Tbx5 expression. The expression of NKX2.5 in terminally differentiated C2C12 myotubes resulted in a change in morphology and breakdown into smaller myotubes. Furthermore, overexpression of NKX2.5 in C2C12 cells and primary cultures of human fetal myoblasts led to differentiation of myoblasts into neuron-like cells and expression of neuronal markers. This study sheds light on the previously unknown non-cardiac functions of Nkx2.5 transcription factor.

Cytokinetic analysis of cell cycle and sub-phases in MOLT-4 cells by cyclin E + A/DNA multiparameter flow cytometry

Cell cycle analysis has become increasingly important in verifying the effect of anti-tumor drugs and cytokinetic research. In the early methods of cell cycle analysis, the flow cytometry relied on DNA content, and therefore, the cell cycle could be only broken into three stages: G(0)/G(1), S, and G(2)/M phase. It could not distinguish the G(0), G(1), G(2), and M phase cells, let alone the sub-phases in G(1) phase. In cell cycle, expression of cyclin E living up to the maximal level in the cells undergoing transition from G(1) to S phase, and G(2) + M cells are cyclin E negative. Expression of cyclin A is progressively increasing during S phase and is maximal in G(2) phase cells. Therefore, in the current study we established a cyclin E + A/DNA multiparameter flow cytometric technique by using a mixture of cyclin E and cyclin A antibodies, which can identify six stages in the whole cell cycle: G(0), early G(1), late G(1), S, G(2), and M phase. Furthermore, we found that cyclin E + A/DNA multiparameter flow cytometry could also be used for stathmokinetic analysis of lymphocyte leukemia MOLT-4 cells after addition of the stathmokinetic agent vinblastine to cultures of exponentially growing MOLT-4 cells. We believe that this new technique will provide a much better tool for molecular cell biology research and especially for cell proliferation kinetics investigations.

Methods for cell cycle analysis and detection of apoptosis of teleost cells.

Flow cytometric techniques have not been previously used on a routine basis to study teleost cell growth and development. In the present chapter, flow instrumentation and cell preparation protocols are given in order to provide evaluation criteria characteristic of different phases of the cell cycle. Flow cytometry is used as an analytical and diagnostic tool to measure DNA ploidy as well as to measure alterations in cell cycle profiles characteristic of random DNA fragmentation (necrosis) compared to patterned DNA cleavage (apoptosis). The types of information obtained by flow analysis include the visualization of cell subpopulations with differing DNA content. For each identified nuclei subpopulation, the parameters of population size, fractions of nuclei in each phase of the cell cycle and computation of DNA ratios can be discerned. Data are presented of ex vivo prepared teleost nonspecific cytotoxic cells (NCC) at resting phase compared to NCC undergoing DNA hypoploid changes characteristic of apoptosis. These cells are compared with a teleost tissue cultured cell line maintained under optimum cell growth conditions versus cells undergoing necrotic cellular pathology. Finally, the requirements for optimum flow analysis are described. Techniques including gating strategies, voltage and gain settings, discrimination options and data collection and interpretation are provided.

Biological characterisation of superficial bladder cancer by bivariate cytokeratin 7/DNA analysis, flow cytometric assessment of MIB- 1, and an immunohistochemical study.

A total of 238 cases of bladder carcinoma stages Ta, Tis, T1 were submitted prospectively to multiparameter flow cytometry and immunohistochemical study in order to determine the biological aggressiveness of the tumour. DNA index (DI), S-phase fraction (SPF) obtained by bivariate cytokeratin 7=DNA analyses, and the immunohistochemical evaluation of p53 and MIB-1 were studied in relation to the traditional prognostic factors in bladder cancer (stage and grade). The variance analysis results showed that DNA aneuploidy was significantly associated with high stage (p = 0:0001), high grade (p = 0:0001), high SPF value ≥5.5% (p = 0:0001), MIB-1 positivity ≥31% (p = 0:0001) and high expression of p53 (staining involving >50% of cells, p = 0:0001). Even if there was no statistical significance the hypotetraploid class (1.70<DI<1.89) showed poor prognostic biomarkers more frequently than the other aneuploid classes. Out of 238 cases, 101 were also submitted to flow cytometric measurement ofMIB-1 (fMIB-1) to study the correlation between cell proliferation and DNA content. Data obtained from fresh, 3 : 1 methanol=acetone fixed samples were compared with values obtained from both cell cycle analysis methods and routine application of the MIB-1 immunostaining in histological sections. fMIB-1 values were positively correlated with SPF values (r = 0:801, p < 0:01) and S+G2M fraction (percentage of cells in S and in G2M phases) (r = 0:763, p < 0:01) but no correlation with paraffin sections was found. A fMIB-1 value >7% was strongly associated with aneuploidy (p = 0:0001). The determination of DNA content coupled with the study of the epithelial (cytokeratin 7) and proliferative (MIB-1) markers could be useful in providing important information on the biological behaviour of superficial bladder tumours.

ACTIVE GROWTH OF THE OCEANIC DINOFLAGELLATE CERATIUM TERES IN THE CARIBBEAN AND SARGASSO SEAS ESTIMATED BY CELL CYCLE ANALYSIS1

ABSTRACTThe growth rate of an oceanic dinoflagellate, Ceratium teres Kofoid, was investigated in the Sargasso and Caribbean Seas from September 1989 to July 1990 using the cell cycle analysis method. Estimated growth rates ranged from 0.29 to 0.58 day−1 and were 1.5–7.2 times higher than generally accepted rates for oceanic dinoflagellates. The higher rates in this report were mainly due to an improvement in techniques that determine the duration of a terminal cell cycle phase in situ. The day‐to‐day variation in growth rates was surprisingly small, but, from long‐term measurements, a weak correlation was found among temperature, daily irradiance, and seasonal growth rate. The calculated species‐specific primary production ranged from 0.5 to 1.8 mg C·m−2·day−1, about 1% of the estimated total production. Ceratium teres may be an important carbon source at the base of the grazing food chain.

Rapid method for cell cycle analysis in a predatory marine dinoflagellate.

Oxyrrhis marina (Dujardin) is a predatory marine dinoflagellate that feeds phagocytically on live phytoplanktonic "prey" cells from the surrounding environment. A rapid method was developed to separate the cell cycle characteristics of these predators from their prey cells in order to study the cell cycle dynamics of this organism. Nuclei from Oxyrrhis were isolated in low salt buffer (PBS) using detergent and mechanical agitation and the DNA stained with Hoechst 33258 in a one step procedure. The method permitted the isolation of nuclei from the Oxyrrhis cells with > 95% efficiency. Discrimination between prey cell nuclei and those of Oxyrrhis was achieved during flow cytometric analysis which yielded routinely G1 CVs of 3-6% for exponentially growing cell populations and 2-3% for stationary phase cells. The method was used to demonstrate the changes in cell cycle dynamics during the exponential and stationary phases of growth. Results indicated that in contrast to most mammalian and phytoplankton cell types Oxyrrhis spent the major portion (ca. 50%) of its cell cycle in G2 + M when actively dividing. Analysis of stationary phase populations also suggests that specific cell cycle control (or restriction) points were present in both G1 and G2 in this species.

An investigation of different methods of cell cycle analysis by flow cytometry in rectal cancer

An investigation of different methods of cell cycle analysis by flow cytometry in rectal cancer