Cancer Cell Apoptosis with FACS Analysis

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Questions and Answers

What is the primary purpose of using FACS analysis in the context presented?

To determine cell growth rates

To analyze nutrient absorption

To measure enzyme activity

To assess cell apoptosis (correct)

What does the hypodiploid peak in the cell cycle histogram represent?

Normal cell division

Cellular differentiation

Senescent cells

Apoptotic cells (correct)

Why might a general laboratory choose not to use flow cytometry?

It requires specialized training only

It is less sensitive than other methods

It is difficult to interpret cell cycle data

It is costly to run and may not be readily accessible (correct)

What factor is indicated by the symbols and * in the context of the drug treatments?

Statistical significance levels Signup and view all the answers

Which cell line was used in the FACS analysis along with MDA-MB-231?

MCF-7 Signup and view all the answers

What is a noted advantage of flow cytometry over AO/EB staining?

Flow cytometry can perform simultaneous cell cycle analysis Signup and view all the answers

What is one of the limitations of using flow cytometry as specified?

Not widely accessible due to costs Signup and view all the answers

What does the term experimental replicates refer to in the context given?

Multiple tests conducted under identical conditions Signup and view all the answers

What is the primary purpose of the clonogenic assay?

To assess a cell's ability to grow into a colony Signup and view all the answers

Which method is considered more economical compared to flow cytometry?

Fluorescent staining Signup and view all the answers

What staining method is used in the colony-forming assay to visualize colonies?

Crystal violet Signup and view all the answers

What is the minimum number of cells required for a colony in a clonogenic assay?

50 cells Signup and view all the answers

Which type of cancer cells were used in the clonogenic assay as referenced?

Colorectal cancer cells Signup and view all the answers

Which treatment is known to reduce the development of colorectal cancer as mentioned in the content?

Phloretin Signup and view all the answers

What condition allows the cells to maintain their morphology during the clonogenic assay?

30%-40% confluency Signup and view all the answers

What is the primary reason for conducting the clonogenic assay after treatment with cytotoxic agents?

To measure reproductive death of cells Signup and view all the answers

What mutation is correlated with the initiation of more than 80% of colorectal cancers?

APC mutation Signup and view all the answers

In the clonogenic assay, what was used to stain the colonies formed by treated HCT-116 cells?

Crystal violet Signup and view all the answers

Which concentration of phloretin resulted in the most significant reduction of colonies in HCT-116 cells?

200 µM Signup and view all the answers

What phase of the cell cycle showed accumulation of cells after treatment with phloretin?

G2/M phase Signup and view all the answers

What statistical significance was observed in the colony count at 200 µM phloretin compared to control cells?

p≤0.0001 Signup and view all the answers

What was the result of the clonogenic assay in terms of colony formation after phloretin treatment?

Significant reduction in colony number Signup and view all the answers

Which assay was used to observe apoptosis in cells treated with phloretin?

Annexin V-FITC/PI assay Signup and view all the answers

What was used for counting colonies formed in the clonogenic assay?

ImageJ software Signup and view all the answers

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.