Flow Cytometry and Cell Differentiation Quiz

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Questions and Answers

Forward scatter channel (FSC) collects data on cell granularity.

False

Fluorescent channels in flow cytometry can provide physical parameters about the cell.

False

Intracellular markers require cell permeabilization to allow antibodies access.

True

In histogram modality, the X-axis represents the number of cells.

False Signup and view all the answers

Side scatter channel (SSC) can provide information about cell size.

False Signup and view all the answers

Cell differentiation results in the same protein expression profile for all cell types.

False Signup and view all the answers

Dot plots represent the overall profile of the cell population.

True Signup and view all the answers

Cell signalling is only involved in cell proliferation and does not affect cell specialization.

False Signup and view all the answers

Live labeling is compatible with both surface and intracellular markers.

False Signup and view all the answers

The first cleavage of a zygote leads to the segregation of determinants and results in identical daughter cells.

False Signup and view all the answers

Flow cytometry can utilize both direct and indirect antibody labeling methods.

True Signup and view all the answers

Morphogens are signaling molecules that can create gradients to induce differentiation in cells.

True Signup and view all the answers

All cells receive the same stimuli during their division, resulting in uniform behavior.

False Signup and view all the answers

The cell lineage model suggests that diversity arises from symmetrical separation of cells.

False Signup and view all the answers

Development is solely based on growth, with no consideration of patterning.

False Signup and view all the answers

Apoptosis is characterized by cell shrinkage and nuclear fragmentation.

True Signup and view all the answers

Cells at the precursor stage acquire diverse fates primarily from external signals.

False Signup and view all the answers

Phosphatidylserine (PS) is found on the outer leaflet of healthy cell membranes.

False Signup and view all the answers

Annexin V is used to detect cells in the late stages of apoptosis.

False Signup and view all the answers

Caspases are one of the two main groups of proteins involved in the mediation of apoptosis.

True Signup and view all the answers

A labeled Annexin V can be used in flow cytometry to detect apoptosis.

True Signup and view all the answers

Propidium iodide (PI) can penetrate the membranes of healthy cells.

False Signup and view all the answers

The Tunel assay is used to measure the absorption of purple light during cell viability assays.

False Signup and view all the answers

The presence of more apoptotic cells indicates increased cell viability.

False Signup and view all the answers

Cells on the front edge and back edge exhibit the same phenotype.

False Signup and view all the answers

Chemotaxis allows cells to move towards a source of a biological signal.

True Signup and view all the answers

Contact inhibition of migration causes cells to keep moving in the same direction after colliding.

False Signup and view all the answers

The leading part of a migrating cell will extend while the tail reduces in size.

True Signup and view all the answers

In a scratch assay, a larger scratch diameter can help improve consistency in results.

False Signup and view all the answers

High cell density stimulates migration while low cell density causes cells to stop migrating.

True Signup and view all the answers

Cytokines and chemokines serve as chemo-attractants during cell migration.

True Signup and view all the answers

The scratch assay is performed to assess the closure rate of a gap created in a cell layer.

True Signup and view all the answers

During PCR, the denaturation step occurs at temperatures below 90°C.

False Signup and view all the answers

Quantitative RT-PCR uses fluorescent tags for real-time detection of amplified fragments.

True Signup and view all the answers

The Ct value indicates the number of DNA copies present at the beginning of the reaction, with a lower Ct suggesting more copies.

True Signup and view all the answers

In RT-PCR, the RNA part is preserved while the DNA part gets digested.

False Signup and view all the answers

The elongation step in PCR occurs at approximately 50°C.

False Signup and view all the answers

The SYBR Green method produces a fluorescent signal only when it binds to single-stranded DNA.

False Signup and view all the answers

Traditional RT-PCR can include a second PCR using a housekeeping control protein for semi-quantitative analysis.

True Signup and view all the answers

Gel electrophoresis is a method used exclusively for detecting amplified RNA fragments during PCR.

False Signup and view all the answers

Microfluidics-based chemotactic migration assays allow cells to migrate towards a medium in a separate micro-well.

True Signup and view all the answers

In a microfluidics-based assay, the negative control contains the same medium with molecules in both micro-wells.

False Signup and view all the answers

The capillary migration assay uses capillaries of the same diameter to analyze cell migration.

False Signup and view all the answers

In a Boyden chamber system, a microporous membrane separates the two chambers filled with medium.

True Signup and view all the answers

Membrane migration assays require the cells to be seeded in the lower compartment of a chamber.

False Signup and view all the answers

Chemoattractants used in invasiveness assays are typically strong chemical signals, such as serum.

True Signup and view all the answers

Trans-well systems have two chambers that are separate from each other and do not include a membrane.

False Signup and view all the answers

In the transmigration assay, the goal is to measure the capacity of cells to migrate through obstacles.

True Signup and view all the answers

What is the significance of telomerase reactivation in cancer cells?

Telomerase reactivation allows cancer cells to replicate indefinitely by maintaining telomere length. Signup and view all the answers

How does quiescence differ from differentiated cell states?

Quiescent cells have stopped dividing but can potentially re-enter the cell cycle, while differentiated cells have permanently lost their capacity to proliferate. Signup and view all the answers

What is the formula used to calculate population doubling time (PDT)?

PDT is calculated using the formula: $\frac{[duration\ of\ experiment \times log(2)]}{[log(C_{final}) - log(C_{initial})]}$. Signup and view all the answers

What role does propidium iodide (PI) play in cell cycle analysis?

Propidium iodide intercalates with double-stranded DNA, allowing researchers to analyze the different phases of the cell cycle based on fluorescence intensity. Signup and view all the answers

What are some physiological factors that can stop cell proliferation?

Physiological factors include metabolic stimuli, confluency, chemical compounds, mechanical stimuli, and cellular aging. Signup and view all the answers

Why can differentiating cells produce more proteins than others in a protein quantification assay?

Differentiated cells often have specialized functions requiring increased protein synthesis, leading to variability in protein content among different cell types. Signup and view all the answers

Describe the significance of analyzing DNA content in cell culture wells using fluorescent dyes.

Analyzing DNA content provides an indirect measure of cell number and enables the assessment of cell cycle phases through fluorescence intensity. Signup and view all the answers

What can live imaging of cells reveal about cell growth?

Live imaging allows for quantification of how much surface area is covered by cells over time, providing insights into growth dynamics. Signup and view all the answers

What is the significance of using different CD markers in flow cytometry?

Different CD markers allow for the discrimination of various cell populations based on their specific surface profiles. Signup and view all the answers

How can flow cytometry be utilized for transfection analysis?

Flow cytometry detects 'positive cells' that have taken in a trans-gene by analyzing the expression of fluorescent proteins. Signup and view all the answers

What are some key controls used in flow cytometry, and why are they important?

Key controls include unstained controls and isotype controls, which help determine autofluorescence and non-specific binding, respectively. Signup and view all the answers

What is the purpose of separating cell populations in FACS?

FACS separates cell populations based on specific criteria such as size and fluorescence to facilitate targeted analysis. Signup and view all the answers

Why is it beneficial to use fluorophores that are far apart in wavelength during flow cytometry?

Using fluorophores with distinct wavelengths enhances the ability to discriminate between signals in the detector. Signup and view all the answers

What types of samples can be analyzed using flow cytometry?

Flow cytometry can analyze biological samples, including stained cells, DNA-binding dyes, and cellular assays. Signup and view all the answers

How does flow cytometry assist in immunophenotyping?

Flow cytometry enables the profiling of cell populations using specific monoclonal antibodies against surface markers. Signup and view all the answers

What role do fluorescent dyes play in flow cytometry beyond protein detection?

Fluorescent dyes can be used to detect lipids and measure cellular components, such as calcium levels. Signup and view all the answers

What is the main advantage of multiplexing in protein analysis?

Multiplexing allows for the simultaneous detection of multiple protein markers in a single sample, increasing efficiency and data richness. Signup and view all the answers

How does high content imaging facilitate the analysis of protein expression in samples?

High content imaging automates the acquisition of fluorescent images and can analyze large-scale datasets efficiently. Signup and view all the answers

What role do appropriate controls play in protein detection methods?

Controls are necessary to validate results and minimize the risk of false positives and false negatives in detection assays. Signup and view all the answers

Explain the difference between quantitative and qualitative protein detection techniques.

Quantitative techniques, like Western blots, measure the amount of target proteins, while qualitative techniques, like immunostaining, assess the presence and distribution of proteins. Signup and view all the answers

Why is flow cytometry considered an effective single-cell detection method?

Flow cytometry analyzes individual cells in a fluid stream, allowing for the characterization of cell populations based on specific markers. Signup and view all the answers

What is the significance of using different fluorochromes in multiplexing?

Different fluorochromes enable the distinct labeling of multiple proteins, facilitating simultaneous analysis of diverse protein interactions. Signup and view all the answers

How do AI and deep learning enhance the analysis of high content imaging data?

AI and deep learning facilitate advanced data processing and statistical analysis, improving accuracy and insights derived from imaging data. Signup and view all the answers

What are the characteristics of automated image acquisition in protein detection?

Automated image acquisition captures fluorescent images consistently and reduces variability in data collection. Signup and view all the answers

How do cells exhibit contact inhibition of migration?

Cells change their migration direction when they collide with similar cells, effectively redirecting their movement. Signup and view all the answers

What is chemotaxis and what role do chemo-attractants play in it?

Chemotaxis is the directed movement of cells toward higher concentrations of biological signals, known as chemo-attractants, such as cytokines and growth factors. Signup and view all the answers

Describe the process and purpose of a scratch assay in studying cell migration.

A scratch assay involves creating a gap in a cell layer and observing how quickly cells migrate to close it, which helps determine migration speed and cellular responses to treatments. Signup and view all the answers

What changes occur in the morphology of a cell during migration in response to a signal?

The cell becomes polarized, extending a protrusion at the front while the tail contracts, which allows it to move toward the source of the signal. Signup and view all the answers

How does cell density influence the migration of cells according to contact stimulation of migration?

Cells in high-density areas will migrate, while those in low-density areas will stop migrating and become isolated. Signup and view all the answers

What limitations are associated with the traditional scratch assay?

Variability in scratch diameter caused by manual scratching can lead to inconsistent results among trials. Signup and view all the answers

Explain how a cell adjusts to maintain its overall volume during migration.

As the leading edge extends forward, the tail of the cell reduces in size to keep the cell's overall volume consistent. Signup and view all the answers

What is the significance of cell polarization in the context of chemotaxis?

Cell polarization is significant as it enables directional movement toward the chemo-attractant, facilitating appropriate responses to environmental signals. Signup and view all the answers

What is a major limitation of SYBR Green in identifying specific types of dsDNA?

SYBR Green cannot discriminate between different types of dsDNA, as it only reflects the total amount of dsDNA present. Signup and view all the answers

How does the TaqMan method achieve specificity in detecting target DNA sequences?

The TaqMan method uses sequence-specific probes that become fluorescent only when the probe is cleaved during DNA synthesis, ensuring detection is specific to the target sequence. Signup and view all the answers

What happens to the fluorescent signal of the TaqMan probe after the polymerase synthesizes a double-stranded copy?

The fluorescent signal is released when the probe is cleaved, separating the reporter from the quencher, which allows the reporter to emit light. Signup and view all the answers

What technique is employed in a Marker Survey for gene expression analysis?

A Marker Survey utilizes gene expression microarrays that analyze mRNA by reverse-transcribing it into labeled cDNA. Signup and view all the answers

Why is it important to use a tag during the reverse transcription of RNA in gene expression analysis?

The tag allows the produced cDNA to be easily identified and quantified during subsequent analysis. Signup and view all the answers

In the context of fluorescence-based DNA quantification, explain the role of the quencher in the TaqMan assay.

The quencher prevents fluorescence when it is close to the reporter, thus maintaining a non-fluorescent state until cleavage occurs. Signup and view all the answers

What is the advantage of using multi-well systems in gene expression analysis?

Multi-well systems allow for the simultaneous analysis of multiple samples, increasing efficiency and throughput in experiments. Signup and view all the answers

How does the design of TaqMan probes contribute to their effectiveness in quantifying specific genes?

TaqMan probes are designed to bind specifically to known target sequences, enabling highly accurate detection of gene expression in quantitative analysis. Signup and view all the answers

What is the purpose of adding a tagged probe to a sample in the ISH technique?

The tagged probe is used to specifically bind to the target nucleotide sequence, allowing for visualization of the target. Signup and view all the answers

Explain the role of negative controls in the RNA ISH experiments.

Negative controls are essential to ensure that false positives are detected, as they use probes that shouldn't bind to the target due to lack of complementarity. Signup and view all the answers

What does FISH stand for, and in what context is it used?

FISH stands for Fluorescence In Situ Hybridization, and it is used to localize specific sequences of DNA in the genome. Signup and view all the answers

Describe the importance of using a pair of probes in the RNAscope technique.

Using a pair of probes ensures that a signal is produced only when both probes are bound to the target RNA, enhancing specificity and accuracy. Signup and view all the answers

What are the limitations of traditional ISH techniques when working with multiple genes?

Traditional ISH techniques become impractical and expensive since each gene requires its own specific probe, making it a challenge for large-scale studies. Signup and view all the answers

How does the tagging method used in probe synthesis affect the detection of targets in ISH?

The tags attached to the probes, whether radioactive, fluorescent, or enzymatic, determine the method for detection and the sensitivity of identifying the target. Signup and view all the answers

What are the potential applications of ISH in understanding gene expression patterns?

ISH can determine which cell types or tissues express specific genes and quantify the number of cells showing that expression. Signup and view all the answers

What is the significance of probe hybridization in the ISH process?

Probe hybridization is crucial as it allows the probe to bind specifically to the complementary target sequence in the sample, facilitating detection. Signup and view all the answers

Study Notes

Cell Signaling Principles

Cell phenotype is defined by the expression of genes into mRNA and protein, with different cell types having different gene expression profiles.
Development involves growth and patterning, controlled by cell signaling between different cell types.
Key cell mechanisms include: cell proliferation, specialization, interaction, and movement.
The zygote (first fertilized egg) has 3 germ layers (mesoderm, endoderm, ectoderm), forming all other tissues.

Generating Cell Diversity

Cells acquire diverse fates through intrinsic cellular information in progenitor cells. The first cleavage results in the segregation of determinants during cell division. As the divisions extend, diversity increases due to uneven distribution of content (mRNA, cytokines, etc).
Cell diversity can be driven by signals from other cells/surrounding, the concentration of signaling molecules (flag model), direct morphogen gradients (cells locally produce an inducer that forms a gradient), and indirect morphogen gradients (localized production of an inhibitor creating a gradient in a distributed inducer).
Patterning via sequential cell induction can result in the formation of many cell types from a few, via interactions between neighboring cells (e.g., zone C cells interacting with cells in zone A and B to influence their neighboring cells).

Different Ways of Transmitting Signals

Signals can be transmitted via diffusion of molecules or electric signals, direct contact between two cells, and gap junctions.
Receptor-mediated signaling involves an extracellular molecule interacting with a receptor protein, triggering intracellular signaling cascades affecting metabolism, gene expression, and/or altering cell shape, leading to signal amplification.
Examples include MAPK pathways.

Signaling Pathways

Wnt pathway (β-catenin): Two main receptors (LRP and Frizzled) are activated by Wnt, releasing β-catenin. The release moves near the nucleus where it modifies transcription of Wnt-responsive genes.
TGF-β Pathway: Two receptors bind to the ligand (BMPs, GDFs...), leading to the phosphorylation cascade of other molecules. The cascade is initiated by Smad2/3 oligomerization with Smad4 which then triggers transcription of target genes. This pathway is important for left-right asymmetry and skeletal formation. BMP signaling often relies on Smad1/5/8 pathways for different outcomes compared to Smad2/3-mediated pathways.
Hedgehog Pathway: Two receptors (patched and smoothened) are bound together in the absence of ligand, while Ci protein (Gli protein in vertebrates) gets bound to microtubules. Ligand binding releases the smoothened protein, influencing PKA and Slimb to trigger a cascade that transcribes responsive genes impacting axis formation and other functions.

Receptor Tyrosine Kinase (RTK) pathway

RTKs involve intracellular tyrosine kinase components and phosphorylation cascades
Examples of RTKs: PDGFR, FGFR, EGFR
FGF molecules often bind to glycosaminoglycan chains, leading to receptor cross-phosphorylation and Ras activation. Ras-Raf-Mek-MAPK pathway then result in activation of genes.
Notch/delta signaling is cell-cell contact-dependent. Delta signals (which are on one cell) will inhibit the target cell. If the target cell receives no signal from delta it will express the Notch receptors on its plasma membrane. Both Notch and Delta must be on the surface. The tail of Notch then migrates to nucleus promoting transcription of target genes. Important for development of nervous system (etc).

Cell Proliferation Analysis

Somatic cell division involves G0, G1, S, G2, and M phases (prophase, metaphase, anaphase, telophase) regulated by CDK/cyclin complexes.
DNA duplicates during the S phase.
Telomeres shorten with each division, potentially stopping cell proliferation.
Cell quiescence is a state where cells stop dividing but can sometimes re-enter the cell cycle.
Physiological factors affecting proliferation include metabolic stimuli, confluence, chemical compounds, mechanical stimuli, and cellular aging.

Cell Cycle Analysis

Propidium iodide (PI) is a red fluorescent DNA stain used for analyzing cell cycle phases by flow cytometry, correlating DNA content with fluorescence intensity.
Cell cycle phases (G0, G1, S, G2, M) are visually discernible via the use of flow cytometry using PI.
Cell cycle analysis assesses cell population proportion in each phase, providing information on proliferation capacity.

Cell Proliferation Assays/Methods

Single measurements: Counting cells using Bürker chambers or automated counters
Population Doubling Time (PDT): Measuring the time it takes for cells to double in number.

Cell Cycle Analysis Techniques

Using propidium iodide (PI) with flow cytometry to measure DNA content and determine cell cycle phase.
Incorporating labeled nucleotides into DNA and measuring the amount over time to determine the rate of cell division and determine phases in a cell cycle.

Proliferation Marker Analysis /Techniques

PCNA (proliferating cell nuclear antigen): found in cells during G1 and S phase; can be absent in resting G0 cells
Ki67: nuclear DNA binding protein; expressed in G1, S, G2, and M phase.
pHH3: phosphorylated histone H3; found in mitotic cells during the M phase.
CFSE (carboxyfluorescein succinimidyl ester - cytoplasmic fluorescent dye). The dye gets diluted in each division, meaning the more divisions there are the smaller the fluorescence is.

Protein Marker Detection Techniques

Proteomics involves systematic characterization of proteins in samples by extracting, digesting proteins to peptides, separating, and analyzing in mass spectroscopy.
Antibody-based techniques (Western blot, dot blot) determine if the target protein is present in a sample.
Immunostaining is useful for localized detection (qualitative signal identification) of protein targets' spatial distribution within a sample.

Multiplexing

Detection of several different protein markers in one sample using different secondary antibodies that are conjugated to different tags; allows co-detection of different fluorochromes in the samples.
Automated image capturing and analysis using image thresholding helps examine multiple well plates in a single experiment/multiple labels on sample wells.

Flow Cytometry Analysis

Flow cytometry measures cell characteristics (size, granularity, fluorescence) as cells pass by a laser stream.
Forward scatter (FSC) measures cell size, and Side scatter (SSC) measures cell granularity, while fluorescent channels measure fluorescence from dyes bound to intracellular or membrane-bound proteins/targets.
Cells can be sorted based on characteristics (via antibodies labeled with fluorophores)
Live-cell experiments possible with some fluorophores Antibody-based labeling and surface/intracellular markers

Cell Migration Analysis

Cell migration is influenced by phenotype and neighboring cells, involving membrane protrusion & cytoskeleton reorganization.
Migration types include leader-follower (cells coordinating), contact inhibition (migration direction change upon cell contact), & contact stimulation (high cell density promotes migration).
Signals (cytokines, chemokines, growth factors) affect cell migration direction (chemotaxis).
The cells become polarized with a higher concentration of attractant at one side of the cell to the other, creating protrusion.
Key elements in migration include adhesion (integrins, actin), intermediary proteins (talin, vinculin), and cytoskeleton rearrangements.
Extracellular matrix proteins (ECM proteins) can affect cell behavior (e.g., surface wettability and topography for cell adhesion & organization).

Cell Migration Assays

Scratch Assay: Observing cells migrating to close a scratched area over time to measure the speed.
Cell-exclusion zone: Measuring migration by creating gaps.
2D Migration Track Monitoring: Tracking cell movement over set time periods.
Flow Chambers: Measuring migration in a controlled microfluidic device.
Impedance Measurement: Measuring the opposition to electrical flow by cells interacting with electrodes over time.

Collective Cell Migration

Cells migrating together (e.g., during tissue development/repair or tumor metastasis)
Mechanisms like leader-follower, contact inhibition/stimulation

Cell Sorting

FACS (Fluorescence-activated cell sorting) separates cells based on characteristics (size, fluorescence)
MACS (Magnetic-activated cell sorting) separates cells based on antibodies bound to magnetic nanoparticles.

Cell Health Assays

Live/Dead Assays assess cell viability by observing whether a dye (e.g. trypan blue) can enter the cell.
Nuclear-ID dual dye method: Using fluorescent dyes (one permeable to live cells, one to dead cells) to quickly quantify dead vs live cells visually and in flow cytometry.
Esterase Activity: Determining cell viability based on the activity of cellular esterases.
ATP Assay: Measuring cellular ATP levels as an indicator of cell viability to assess viable cells in a sample (it is sensitive and non-destructive).
MTT Assay: measuring the amount of viable cells, as an indicator of cell viability by observing colorimetric changes.
LDH Assay: Measuring lactate dehydrogenase (LDH) release from dead cells as a measure of cell viability.

Apoptosis Assays

Phosphatidylserine (PS) assay/Annexin V-FITC: Measuring PS redistribution to the outer leaflet of the cell membrane, a hallmark of apoptosis, using fluorescently labeled Annexin V binding to PS.
TUNEL Assay (Terminal deoxynucleotidyl transferase dUTP nick-end labeling): Identifying DNA fragmentation as a result of degradation from damage or apoptosis using an enzymatic reaction to add a fluorescent tag.
Caspase Assays: Measuring caspase activity, as activated caspases participate in apoptosis.

Cell Viability Assays - Cell Senescence

SA-β-gal (Senescence-associated β-galactosidase). Observing blue staining due to β-gal activity as a marker of senescence.
Confocal Raman Microscopy (CRM). Analyzing structural differences between live and dead cells.
Raman Spectroscopy. A technique for identifying different biochemical molecules. Based on the use of a laser onto a sample and detecting the scattered light at shifted frequencies which are unique to each compound present and that can be used to distinguish healthy cells from dead/cancer cells.

Gene Expression Analysis

Northern Blotting: Detecting specific RNA molecules in a sample through their hybridisation with a labeled DNA probe.
RT-PCR (Reverse Transcriptase PCR): Converting RNA to cDNA, then amplifying it to determine gene expression (quantitative).
Quantitative RT-PCR (RT-qPCR): Measuring gene expression in real-time during PCR to detect gene expression in a quantitative manner.
Microarray Analysis: Using arrays with probes to detect mRNA expressed in the samples of interest (e.g., different tumor tissues or different cells).

RNA Markers - Spatial Localized Detection

Laser Capture Microdissection (LCM): Isolating specific regions/cells in a sample to analyse the RNA markers associated in a subset of the sample (spatial).
In Situ Hybridization (ISH): Detecting the presence of specific RNA sequences in cells within a tissue (spatial, qualitative).
RNAscope: A multiplex target survey for RNA. Single RNA molecules in tissue/cell samples are marked with fluorescent tags/barcodes, enabling the detection of multiple transcripts at the same time (multiplexing).
Spatial Molecular Imaging: Similar to the RNAscope method, but it detects transcripts or protein tags as well via barcode tagging allowing for multiple transcripts/targets/markers.

Single-Cell RNA Sequencing (scRNA-seq)

Isolating single cells
Preparing RNA
Sequencing RNA, mapping to a known genome to determine gene expression
Data analysis

Cell Engineering

Inducing gene expression: Introducing a foreign gene usually by using strong promoters, enabling a target protein production and protein extraction analyses.
Repressing gene expression: Silencing gene expression using RNA interference (RNAi) or taking the sequence out of the genome or introducing mutations.
Triggering mutations: Changing the protein sequence by introducing point mutations

Gene Editing

Knockout: Deleting or modifying a gene's function.
Homology-directed Repair (HDR): Precisely inserting a new DNA fragment into a specific genomic location.
Non-homologous End Joining (NHEJ): Repairing DNA breaks randomly, leading to deletions or insertions.
CRISPR-Cas9: A precise genome editing tool that uses a guide RNA to target a specific DNA sequence, enabling precise gene editing.

Cell Culture

Culture cells in controlled environment, sterile conditions
Use sterile equipment, e.g., cell culture-hoods and laminar flow
CO2 incubators: Maintain proper temperature, humidity, and CO2 levels.
Basal media (e.g. DMEM, RPMI, and Ham's F12) with essential substances (serum, salts, nutrients, amino acids, etc.)

Cell Detachment Techniques

Mechanical Detachment: Scrapers
Enzymatic Detachment: Using enzymes (trypsin) to cleave cell-cell adhesion proteins
EDTA-based detachment: Using EDTA to chelate divalent cations involved in binding to cell adhesion

Cell Contamination

Bacteria, yeast, fungi, and mycoplasma are common contaminants that could be recognized via microscopy/pH change/color change.
Risk of cross-contamination is high when handling multiple cell cultures simultaneously; careful procedures (e.g., one-cell-culture-per-time) and sterile laboratory tools needed to avoid this.

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Test your knowledge on flow cytometry, cell granularity, and the intricacies of cell differentiation. This quiz covers topics such as forward and side scatter channels, intracellular markers, and the role of morphogens in signaling. Challenge yourself with questions that assess your understanding of these essential concepts in cell biology.

Flow Cytometry and Cell Differentiation Quiz

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Questions and Answers

Forward scatter channel (FSC) collects data on cell granularity.

Fluorescent channels in flow cytometry can provide physical parameters about the cell.

Intracellular markers require cell permeabilization to allow antibodies access.

In histogram modality, the X-axis represents the number of cells.

Side scatter channel (SSC) can provide information about cell size.

Cell differentiation results in the same protein expression profile for all cell types.

Dot plots represent the overall profile of the cell population.

Cell signalling is only involved in cell proliferation and does not affect cell specialization.

Live labeling is compatible with both surface and intracellular markers.

The first cleavage of a zygote leads to the segregation of determinants and results in identical daughter cells.

Flow cytometry can utilize both direct and indirect antibody labeling methods.

Morphogens are signaling molecules that can create gradients to induce differentiation in cells.

All cells receive the same stimuli during their division, resulting in uniform behavior.

The cell lineage model suggests that diversity arises from symmetrical separation of cells.

Development is solely based on growth, with no consideration of patterning.

Apoptosis is characterized by cell shrinkage and nuclear fragmentation.

Cells at the precursor stage acquire diverse fates primarily from external signals.

Phosphatidylserine (PS) is found on the outer leaflet of healthy cell membranes.

Annexin V is used to detect cells in the late stages of apoptosis.

Caspases are one of the two main groups of proteins involved in the mediation of apoptosis.

A labeled Annexin V can be used in flow cytometry to detect apoptosis.

Propidium iodide (PI) can penetrate the membranes of healthy cells.

The Tunel assay is used to measure the absorption of purple light during cell viability assays.

The presence of more apoptotic cells indicates increased cell viability.

Cells on the front edge and back edge exhibit the same phenotype.

Chemotaxis allows cells to move towards a source of a biological signal.

Contact inhibition of migration causes cells to keep moving in the same direction after colliding.

The leading part of a migrating cell will extend while the tail reduces in size.

In a scratch assay, a larger scratch diameter can help improve consistency in results.

High cell density stimulates migration while low cell density causes cells to stop migrating.

Cytokines and chemokines serve as chemo-attractants during cell migration.

The scratch assay is performed to assess the closure rate of a gap created in a cell layer.

During PCR, the denaturation step occurs at temperatures below 90°C.

Quantitative RT-PCR uses fluorescent tags for real-time detection of amplified fragments.

The Ct value indicates the number of DNA copies present at the beginning of the reaction, with a lower Ct suggesting more copies.

In RT-PCR, the RNA part is preserved while the DNA part gets digested.

The elongation step in PCR occurs at approximately 50°C.

The SYBR Green method produces a fluorescent signal only when it binds to single-stranded DNA.

Traditional RT-PCR can include a second PCR using a housekeeping control protein for semi-quantitative analysis.

Gel electrophoresis is a method used exclusively for detecting amplified RNA fragments during PCR.

Microfluidics-based chemotactic migration assays allow cells to migrate towards a medium in a separate micro-well.

In a microfluidics-based assay, the negative control contains the same medium with molecules in both micro-wells.

The capillary migration assay uses capillaries of the same diameter to analyze cell migration.

In a Boyden chamber system, a microporous membrane separates the two chambers filled with medium.

Membrane migration assays require the cells to be seeded in the lower compartment of a chamber.

Chemoattractants used in invasiveness assays are typically strong chemical signals, such as serum.

Trans-well systems have two chambers that are separate from each other and do not include a membrane.

In the transmigration assay, the goal is to measure the capacity of cells to migrate through obstacles.

What is the significance of telomerase reactivation in cancer cells?

How does quiescence differ from differentiated cell states?

What is the formula used to calculate population doubling time (PDT)?

What role does propidium iodide (PI) play in cell cycle analysis?

What are some physiological factors that can stop cell proliferation?

Why can differentiating cells produce more proteins than others in a protein quantification assay?

Describe the significance of analyzing DNA content in cell culture wells using fluorescent dyes.

What can live imaging of cells reveal about cell growth?

What is the significance of using different CD markers in flow cytometry?

How can flow cytometry be utilized for transfection analysis?

What are some key controls used in flow cytometry, and why are they important?

What is the purpose of separating cell populations in FACS?

Why is it beneficial to use fluorophores that are far apart in wavelength during flow cytometry?

What types of samples can be analyzed using flow cytometry?

How does flow cytometry assist in immunophenotyping?

What role do fluorescent dyes play in flow cytometry beyond protein detection?

What is the main advantage of multiplexing in protein analysis?

How does high content imaging facilitate the analysis of protein expression in samples?

What role do appropriate controls play in protein detection methods?

Explain the difference between quantitative and qualitative protein detection techniques.

Why is flow cytometry considered an effective single-cell detection method?

What is the significance of using different fluorochromes in multiplexing?

How do AI and deep learning enhance the analysis of high content imaging data?

What are the characteristics of automated image acquisition in protein detection?

How do cells exhibit contact inhibition of migration?

What is chemotaxis and what role do chemo-attractants play in it?

Describe the process and purpose of a scratch assay in studying cell migration.

What changes occur in the morphology of a cell during migration in response to a signal?