Podcast
Questions and Answers
What is the primary purpose of using FACS analysis in the context presented?
What does the hypodiploid peak in the cell cycle histogram represent?
Why might a general laboratory choose not to use flow cytometry?
What factor is indicated by the symbols ** and *** in the context of the drug treatments?
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Which cell line was used in the FACS analysis along with MDA-MB-231?
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What is a noted advantage of flow cytometry over AO/EB staining?
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What is one of the limitations of using flow cytometry as specified?
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What does the term experimental replicates refer to in the context given?
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What is the primary purpose of the clonogenic assay?
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Which method is considered more economical compared to flow cytometry?
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What staining method is used in the colony-forming assay to visualize colonies?
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What is the minimum number of cells required for a colony in a clonogenic assay?
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Which type of cancer cells were used in the clonogenic assay as referenced?
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Which treatment is known to reduce the development of colorectal cancer as mentioned in the content?
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What condition allows the cells to maintain their morphology during the clonogenic assay?
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What is the primary reason for conducting the clonogenic assay after treatment with cytotoxic agents?
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What mutation is correlated with the initiation of more than 80% of colorectal cancers?
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In the clonogenic assay, what was used to stain the colonies formed by treated HCT-116 cells?
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Which concentration of phloretin resulted in the most significant reduction of colonies in HCT-116 cells?
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What phase of the cell cycle showed accumulation of cells after treatment with phloretin?
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What statistical significance was observed in the colony count at 200 µM phloretin compared to control cells?
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What was the result of the clonogenic assay in terms of colony formation after phloretin treatment?
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Which assay was used to observe apoptosis in cells treated with phloretin?
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What was used for counting colonies formed in the clonogenic assay?
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Study Notes
Lecture 16 Summary
- Lecture presented by Dr. Kavitha Thirumurugan, Professor HAG, SBST at VIT Vellore Institute of Technology.
- Lecture focused on Fluorescence-activated cell sorting (FACS) analysis of apoptosis in cancer cells.
Annexin V Apoptosis Assay (MCF-7 Cells)
- Annexin V Apoptosis assay using FACS analysis on MCF-7 breast cancer cells.
- Cells treated with different concentrations of SFN, WA, or both compounds.
- Treatment was for 3 days.
- Results show significant apoptosis at higher concentrations (p<0.01, p<0.001)
- Data represents three replicates.
- X-axis displays compound concentration (µM).
Annexin V Apoptosis Assay (MDA-MB-231 Cells)
- Annexin V Apoptosis assay, using FACS on MDA-MB-231 breast cancer cells.
- Cells treated with different concentrations of SFN, WA, or both compounds.
- Treatment was for 3 days.
- Results show significant apoptosis at higher concentrations (p<0.01, p<0.001).
- Data represents three replicates.
- X-axis displays compound concentration (µM).
Flow Cytometry Analysis
- Flow cytometry used by the lecture to analyze the cell cycle in relation to apoptosis.
- Negative control group showed no distinct apoptotic peak.
- Experimental group (undergoing apoptosis) showed an apoptotic peak (Ap peak) before the G1 peak in the histogram.
Flow Cytometry as Method to Detect Apoptosis
- Flow cytometry is a high-sensitivity method for detecting apoptosis.
- It can be used to study the cell cycle simultaneously.
- General laboratories may not have the required equipment (flow cytometer).
- Cost associated with the equipment might be a factor.
- AO/EB staining may be a viable alternative.
Clonogenic Assay
- Clonogenic or Colony Forming Assay is an in vitro technique used to quantify cells capacity to grow into a colony.
- Defined by colonies of at least 50 cells.
- Assesses every cell's ability to undergo uncontrolled division.
Clonogenic Assay as method to determine Cell Reproductive Death
- Clonogenic assay is a critical method for determining cell reproductive death following treatment with ionizing radiation, or other cytotoxins.
- Only a fraction of seeded cells can form colonies.
- Colonies are developed in 1-3 weeks.
- Colonies fixed using glutaraldehyde and stained using crystal violet, then counted with a stereomicroscope.
HCT116 Colony Forming Assay Protocol
- Protocol for assessing the effects of drug treatment on HCT116 colorectal cancer cells.
- Seeding density of 10000 cells per well.
- Cells allowed to reach 30-40% confluency (before treatment)
- Treatment with specific drug concentrations in media.
- Incubation for 3 days (at 37°C, 5% CO2).
- Fixation and staining with Coomassie Blue stain after treatment.
Clonogenic Assay of HCT116 Cells (Treatment with Phloretin)
- HCT116 colorectal cancer cells are treated with phloretin (at various concentrations).
- Phloretin, a dihydrochalcone polyphenol, is found in apples, pears, strawberries.
- Phloretin inhibits CRC development by acting on glucose transporters, and activating p53.
- Over 80% of CRC are initiated by APC (adenomatous polyposis coli) mutations linked with Wnt activation.
Clonogenic assay of HCT116 cells: Image Analysis with Phloretin
- Image analysis was used to quantify the growth of HCT-116 cells after treatment with various concentrations of phloretin.
- The analysis revealed a reduction in the number of colonies as concentrations increased.
- Significant reduction in colony formation at higher concentrations (p<0.05, to p<0.0001).
Clonogenic Assay: Treatment Results and Colony Reduction with Phloretin
- Treatment with phloretin significantly reduced the number of colonies formed in HCT-116 cells (compared to control).
- This reduction was significant (p<0.0001) at the highest concentration of phloretin used.
- Demonstrates Inhibitory effect of phloretin on CRC proliferation
Flow Cytometry Results (HCT-116): Treatment with Phloretin Analysis
- Flow cytometry was employed to investigate the mechanism by which phloretin inhibits cell proliferation in HCT-116 cells.
- There was altered pattern in cell cycle distribution following treatment (increased G2/M phase accumulation).
- Accumulation in G2/M phase showed significant increase (p<0.0001)
Apoptosis Results (HCT-116): Phloretin treatment Analysis
- Assessing the percentage of early- and late-apoptotic cells in HCT-116 cells after phloretin treatment.
- Early and late apoptotic cells significantly increased with higher phloretin concentrations.
- This increase suggests phloretin treatment induces apoptosis in a concentration dependent manner.
Summary of Phloretin's Effect
- Phloretin significantly inhibits HCT-116 cell proliferation.
- Phloretin induces apoptosis in HCT-116 cells in a concentration dependent manner.
Additional experiments
- Further analysis was performed in HCT-116 and SW-480 cells to investigate phloretin-induced apoptosis, showing the significant increase of early and late apoptosis at higher concentrations.
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Description
This quiz delves into the fluorescence-activated cell sorting (FACS) analysis of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. You will explore the effects of different concentrations of SFN and WA compounds on apoptosis rates over a three-day treatment period. Test your knowledge on the methodology and findings of the Annexin V Apoptosis assay.
