Determination of cell cycle parameters by flow cytometry in bacteria: practical considerations

2010 ◽  
Author(s):  
F. Molina ◽  
M. Mota ◽  
M.A. Sánchez-Romero ◽  
A. Jiménez-Sánchez
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry

scholarly journals P04.05 The Effects and Mechanism Involved in Paclitaxel combined with Shikonin on U87 Glioma Cells

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii29-iii29
Author(s):  
J SHI
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Migration ◽  
Inhibitory Effect ◽  
Transwell Assay ◽  
Significant Inhibitory Effect ◽  
Rna Content ◽  
Paclitaxel Group ◽  
U87 Cells

Abstract BACKGROUND Glioma is the most common intracranial primary tumor,chemotherapy seems to be the main treatment for brain glioma.Chemotherapy is currently widely used in clinical treatment of gliomas,however,drug-resistant always appears,so combination of chemotherapy has became an inevitable choice. Paclitaxel and shikonin are stable and efficient chemotherapy drugs which can promote apoptosis and ininhibite the growth of tumor.Recent research reports,paclitaxel and shikonin can respectively activate and inhibit the expression of p-ERK.The purpose of this study was to investigate whether we can inhibite the activation of p-ERK caused by paclitaxel to enhance the effect of paclitaxel,furthermore use the combination of the two drugs to reach a better effect of chemotherapy. MATERIAL AND METHODS 1.Cell Culture2. Drugs Administration.3. Growth Inhibition Assays (CCK-8) .4.Scratch test to observe cell migration 5.Transwell assay for the determination of cell migration 6.Transwell assay for the determination of cell invasion 7. Annexin V-FITC and PI Assay for Flow Cytometry to Detect Apoptosis 8. PI Assay for Flow Cytometry to Detect Cell Cycle 9. Western Blot to Detect the Expression of P-ERK/ERK 10. Statistical Analysis. RESULTS 1.The results tested by CCK-8 showed these two drugs have synergic effects with a CDI equals to 0.72 which means these two drugs are significant synergistic.2.Compared with paclitaxel group or shikonin group,the 1/2paclitaxel+1/2shikonin group had a significant inhibitory effect upon the migration of U87 cells.3.Compared with paclitaxel group and shikonin group,the 1/2paclitaxel+1/2shikonin group had a significant inhibitory effect upon the invation of U87 cells.4.Compared with paclitaxel group and shikonin group,the 1/2paclitaxel+1/2shikonin group had a significant promoting effect upon inducing apoptosis of U87 cells.5. The 1/2paclitaxel+1/2shikonin group significantly improved the apoptosis rate of U87 cells,the RNA content of the G1-phase was significantly increased in shikonin group, the RNA content of the G2- phase significantly increased in paclitaxel group and 1/2paclitaxel+1/2shikonin group.6.Compared with control group,the rate of p-ERK/ERK was increased in paclitaxel group(P<0.05),while the rate of p-ERK/ERK was significantly decreased in the shikonin group and 1/2paclitaxel+1/2shikonin group(P<0.05). CONCLUSION 1. Compared with paclitaxel group and shikonin group,the combination group with half dosage can make synergic effects and significantly inhibit the proliferation,migration and invation ability of U87 cells,besides promote apoptosis of U87 cells and block U87 cell cycle.2. Paclitaxel combined with shikonin could decrease the expression of p-ERK/ERK,which may be involved in the synergic effects of these two drugs.

Myricanol-9-acetate, a novel naturally occurring derivative of myricanol, induces ROS-dependent mitochondrial-mediated Apoptosis in MCF-7 cancer cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Gazanfar Ahmad ◽  
Sameer Ahmad Mir ◽  
Loveleena Kour Anand ◽  
Faheem Hyder Pottoo ◽  
Neerupma Dhiman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Ros Generation ◽  
Cycle Arrest ◽  
Naturally Occurring ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Mcf 7

Background: Low therapeutic efficacy and drug-induced systemic toxicity of currently used anti-cancerous chemotherapeutic agents are major compelling factors for finding out clinically efficient molecules with high efficiency and less toxicity. Objective: The current research work was undertaken to evaluate the anticancer potential of Myricanol-9-acetate (MA), a novel naturally occurring derivative of myricanol. Methods: MCF-7, MiaPaCa-2, and HCT 116 were used for cytotoxicity determination of the MA and ML (Myricanol) by MTT assay. The mechanistic study involved the determination of cell cycle arrest, ΔΨm loss, ROS generation, western blot assay, and flow cytometry by reported methods on MCF-7 cells. Results: MA exhibited anticancer activity against all three cell lines; however, the molecule was found most active against the MCF-7 cell line. We observed IC50 20μM with MA treatment as compared to the IC50 of 42 μM for myricanol treatment. Detailed mechanistic studies revealed that MA induced apoptosis on MCF-7 cell line through ROS generation; a dose-dependent drop in mitochondrial membrane potential was found to be associated with cell cycle arrest at G0/G1 phase. Our results further demonstrated down-regulation of Bcl2 and activation of the caspase cascade as the events involved in the MA-induced apoptosis. Flow cytometry results indicated an increase in early and late apoptotic population in a dose-dependent manner with an apoptotic population of about 20% at 30 μM of MA, thus supporting our results. Conclusion: Present findings thus suggest that MA might serve as a promising novel drug candidate having high scope for further evaluation in preclinical and clinical studies.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

scholarly journals The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies

2021 ◽  
Vol 4 (1) ◽  
pp. 11
Author(s):  
Evrysthenis Vartholomatos ◽  
George Vartholomatos ◽  
George A. Alexiou ◽  
Georgios S. Markopoulos
Keyword(s):  
Central Nervous System ◽  
Flow Cytometry ◽  
Nervous System ◽  
Therapeutic Agents ◽  
Phenotypic Characterization ◽  
Cell Phenotype ◽  
Analytical Technique ◽  
The Past ◽  
Current Review

Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Development of multiparametric analysis of human PBMC by flow cytometry: Determination of inflammatory and calcic profile

2021 ◽  
Vol 13 (2) ◽  
pp. 199-200
Author(s):  
C. Brun ◽  
A. Paccalet ◽  
T. Bochaton ◽  
M. Paillard ◽  
C. Crola Da Silva
Keyword(s):  
Flow Cytometry ◽  
Human Pbmc ◽  
Multiparametric Analysis
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