Cell Biology: Cell Cycle and Apoptosis

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Questions and Answers

What is the main purpose of using the TUNEL detection method?

To quantify normal cell metabolism.

To measure the growth rate of cell populations.

To visualize all cellular components.

To detect apoptotic cells by identifying DNA fragmentation patterns. (correct)

What type of DNA fragmentation is primarily associated with apoptosis according to the described assay?

Linear DNA of varying sizes.

Large intact DNA molecules.

Random DNA fragmentation without a size range.

Fragmentation primarily in 200-250 and 30-50 kilobases. (correct)

Which component is added to the DNA fragments during the TUNEL assay to label them?

DUTP without any labeling.

A radioactive isotope for detection.

Fluorescein isothiocyanate (FITC).

Bromodeoxyuridine triphosphates (Br-dUTP). (correct)

What advantage does the two-step labeling method have over the single-step labeling method in the TUNEL assay?

It provides a more sensitive and stronger signal. Signup and view all the answers

What role does exogenous Terminal deoxynucleotidyl transferase (TdT) play in the TUNEL detection method?

It facilitates the labeling of fragmented DNA. Signup and view all the answers

What is the primary purpose of cell proliferation in tissue homeostasis?

To facilitate cell growth and division Signup and view all the answers

Which of the following correctly identifies the stages of interphase in the cell cycle?

G1, S, G2 Signup and view all the answers

In flow cytometry, what is the role of propidium iodide (PI)?

To quantitatively stain DNA Signup and view all the answers

What happens during the G1 phase of the cell cycle?

Cells monitor their environment Signup and view all the answers

How is apoptosis defined in the context of cell biology?

A programmed self-destruction process Signup and view all the answers

What is the main advantage of using multicolor flow cytometry in studying cell processes?

It can simultaneously monitor various cellular events Signup and view all the answers

What defines the mitotic phase of the cell cycle?

Mother cell divides into daughter cells Signup and view all the answers

Which statement best describes the relationship between apoptosis and cell proliferation?

Balanced apoptosis is essential for normal tissue homeostasis Signup and view all the answers

What characteristic change occurs in the amount of dye in a cell with each replication event?

The amount of dye decreases. Signup and view all the answers

During which phase of the cell cycle is BrdU incorporated into newly synthesized DNA?

S phase Signup and view all the answers

Which antigen is specifically expressed in the nucleus of dividing cells and is not detected in the G0 phase?

Ki67 Signup and view all the answers

What is the primary purpose of the MTT-based assay in studying cell proliferation?

To determine mitochondrial activity of metabolically active cells. Signup and view all the answers

Which of the following is NOT a characteristic change associated with apoptosis?

Formation of cytoplasmic granules Signup and view all the answers

Which technique can be combined with Ki67 to enhance the confidence of detecting cell proliferation?

BrdU Signup and view all the answers

Which assay is used to detect apoptotic cells based on changes like mitochondrial membrane potential?

Annexin V staining Signup and view all the answers

What outcome do the biological and morphological changes during apoptosis influence?

Programmed cell death Signup and view all the answers

What characterizes the fluorescence intensity of cells during the cell cycle?

It correlates with the amount of DNA present in the cells. Signup and view all the answers

What is the primary reason for permeabilizing cells during the cell cycle analysis?

To allow fluorescent dyes to enter the cell. Signup and view all the answers

What role does RNase A play during the staining of cells in the cell cycle analysis?

It removes RNA to avoid staining artefacts. Signup and view all the answers

How can the cell phases be distinguished during flow cytometry?

By measuring the fluorescence intensity of the dyes associated with DNA. Signup and view all the answers

What characteristic of Violet Proliferation Dye 450 (VPD450) allows it to be utilized in flow cytometry?

Its esterified form is nonfluorescent until cleaved by esterases. Signup and view all the answers

What does an increase in cells in the pre-G phase indicate in cell cycle analysis?

Increased cell death compared to control. Signup and view all the answers

Which of the following dyes is typically used in cell cycle analysis for quantifying DNA content?

Propidium Iodide Signup and view all the answers

What indicates the presence of cells in the S phase of the cell cycle during flow cytometry?

Cells with greater than 2n DNA content. Signup and view all the answers

What indicates that a cell is non-apoptotic during the staining process?

Annexin-V negative and PI negative Signup and view all the answers

Which of the following statements about the Annexin V-PI dual staining technique is correct?

It distinguishes early apoptotic cells from necrotic cells. Signup and view all the answers

What is the significance of DNA fragmentation in apoptotic cells?

It is a hallmark of apoptosis. Signup and view all the answers

Which of the following correctly describes the role of propidium iodide (PI) in the staining process?

It differentiates early apoptotic cells from necrotic cells. Signup and view all the answers

What distinguishes late apoptotic cells from necrotic cells in the context of staining?

Both are positive for Annexin-V and PI. Signup and view all the answers

How long does the DNA fragmentation detection method take compared to traditional methods?

It takes less than 90 minutes. Signup and view all the answers

What is indicated by a cell being Annexin-V positive and PI negative?

The cell is in early stages of apoptosis. Signup and view all the answers

Which aspect does the Apoptotic DNA Ladder Detection Kit primarily assess?

The level of DNA fragmentation. Signup and view all the answers

What is the primary mechanism by which the Annexin-V binding assay distinguishes apoptotic cells from non-apoptotic cells?

Relocation of phosphatidylserine to the outer cell membrane Signup and view all the answers

Which of the following statements about phospholipid distribution in viable cells is true?

Choline-containing phospholipids reside mainly on the outer leaflet Signup and view all the answers

What event allows Annexin-V to enter cells during late apoptosis?

Membrane permeabilization Signup and view all the answers

What does a cell stained negative for FITC Annexin V and negative for propidium iodide (PI) indicate?

The cell is viable and healthy Signup and view all the answers

Which fluorescent dye is used alongside Annexin-V in apoptosis assays to evaluate membrane integrity?

Propidium iodide (PI) Signup and view all the answers

What is the approximate molecular weight range of Annexin-V as a phospholipid binding protein?

35-36 kDa Signup and view all the answers

What does the translocation of phosphatidylserine to the outer leaflet signify during apoptosis?

Initiation of the apoptosis pathway Signup and view all the answers

What characteristic of viable cells preserves their phospholipid distribution with phosphatidylserine on the inner leaflet?

Asymmetric distribution of phospholipids Signup and view all the answers

Study Notes

Cell Biology: Cell Cycle Analysis, Proliferation, and Apoptosis

Cell proliferation and apoptosis are crucial for normal tissue homeostasis
Cell proliferation is the increase in cell number due to growth and division, regulated by the cell cycle
Apoptosis, or programmed cell death, results in controlled self-destruction
Methods exist to assess apoptosis, the cell cycle, and cell proliferation, from basic research to drug screening
Multicolor flow cytometry is essential for studying apoptosis, cell cycle, and proliferation
It monitors these processes along with other cellular events, like protein phosphorylation and cytokine secretion, within heterogeneous cell populations

The Cell Cycle

The cell cycle has two major phases: interphase and the mitotic phase
Interphase has three stages: G1, S, and G2
In G1, cells monitor their environment and synthesize RNA and proteins for growth
In S phase, cells replicate DNA
In G2, cells continue to grow and prepare for mitosis

Cell Cycle Analysis Using Flow Cytometry

Flow cytometry is a common method for assessing the cell cycle
It measures cellular DNA content, allowing analysis of different treatments' effects on various cell cycle stages
Cells are permeabilized and stained with a fluorescent dye (e.g., propidium iodide) that stains DNA
A laser measures the fluorescence intensity of each cell, correlating with the amount of DNA
DNA content increases during the S phase
The G2/M phase has twice the fluorescence of the G0/G1 phase

Experimental Procedure for Flow Cytometry

Cells are permeabilized to allow dye entry
Cells are usually treated with RNase A to remove RNAs
Dyes like propidium iodide, DRAQ5, or Hoechst 33342 are used
The flow cytometer measures fluorescence from the dye, correlating with DNA content

Tools and Techniques to Study Cell Proliferation

Violet Proliferation Dye 450 (VPD450) is a nonfluorescent dye that becomes fluorescent inside the cell as the cell replicates
Bromodeoxyuridine (BrdU) is an analog of thymidine; it's incorporated into newly synthesized DNA and can be readily detected
Cell proliferation involves increases in certain proteins like Ki67—expressed only during cell division

Tools and Techniques to Study Apoptosis

Apoptosis is essential for physiological and pathological conditions including embryo/organ development and immune responses
Apoptosis detection is crucial for research and drug development
Apoptosis is characterized by mitochondrial membrane potential changes, caspase activation, DNA fragmentation, and membrane blebbing
Methods like Annexin-V and TUNEL are used to detect apoptosis

Apoptosis Detection Methods (Annexin-V)

Annexin V is a protein that binds to phosphatidylserine, which relocates to the outer leaflet of the plasma membrane during apoptosis
Annexin-V binding is used to differentiate apoptotic cells(positive) from healthy/viable cells (negative)
The method also uses propidium iodide (PI) to distinguish between early apoptosis, late apoptosis/necrosis, or viable cells for a more complete picture

Apoptosis Detection Methods (TUNEL)

TUNEL (Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling) is used to detect DNA fragmentation in apoptosis.
The method labels fragmented DNA with a fluorescent marker
Cell death is determined by comparing cells in the pre-G phase to control cells

Apoptosis Detection Methods (DNA Fragmentation)

Apoptosis is characterized by DNA fragmentation into smaller fragments
This fragmentation is typically visualized by analyzing DNA samples via electrophoresis
Cells with fragmented DNA exhibit a characteristic "DNA ladder" pattern on electrophoresis gels, distinguishing them from non-apoptotic ones

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Description

This quiz covers essential concepts in cell biology, focusing on cell cycle analysis, proliferation, and apoptosis. Participants will explore the phases of the cell cycle, including interphase and mitotic phases, and the methods used to assess these processes. Topics include multicolor flow cytometry and the importance of apoptosis in tissue homeostasis.