Week 3 Flashcards
What is lineage tracing?
- Studies cell ancestry and cell fate.
-a single cell is marked in a way that its progeny can be traced as it divides
What are the 4 important properties of the labelling tracers?
1.it should not chang^e t^he properties of the labelled cell, its progeny or neighboring cells
2. Must be retained over time
3.must be passed onto all progeny of the founder cell(original cell with tracer )
4.must never transfer to unrelated neighboring cells
Isolation of stem cells
-stem cells need to be isolated from a highly heterogenous population
-95% - 100% purity level needs to be achieved to give more accuracy
What is fluorescence activated cell sorting ( FACS )
- electrical charges are placed on cells to break up stream of fluid that the cell is passing through (left, right and middle) so that the cells can be sorted based on what is being targetted
- basically narrows down cells of interest by depositing cells into small wells after they are analyzed based on the criteria
- this method is fast and can look for different things
- this method can still lead to some heterogeneity but index sorting helps with this by narrowing down the criteria being looked for in the cells to create more accuracy
What are the common approaches used to identify stem cells?
- Surface biomarkers
- Metabolic activities
- Cell cycle activity (or lack thereof)
What are the two naming conventions for stem cell surface proteins?
- Gene name – Describes origin, function, and structure.
- Designation – Recognized by a specific antibody.
How can surface molecules be exploited for stem cell isolation?
Stem cells express unique surface proteins that can be targeted using antibodies for selection and isolation.
What are the three key features of flow cytometry?
- Fluidics – Controls sample movement.
- Optics – Uses lasers for detection.
- Electronics – Analyzes emitted signals.
How does flow cytometry help analyze stem cells?
It provides information on protein presence, detecting both surface and internal proteins (if cells are fixed).
What are ABC transporters, and why are they important in stem cells?
ABC transporters are transmembrane proteins involved in expelling diverse chemicals, including toxic substances. ABC-G2 is highly expressed in stem cells and helps them export chemical dyes.
What is the ‘side population’ (SP) phenotype in flow cytometry?
SP cells are those that actively pump out dyes, exhibiting low red and blue light intensity. The SP phenotype is often enriched in stem cells.
How does the calcium channel blocker Verapamil affect the SP phenotype?
Verapamil inhibits dye efflux, reducing the number of cells in the SP, confirming the role of ABC transporters in stem cell function.
How can metabolic features be used to identify stem cells?
- Aldehyde dehydrogenase (ALDH) activity can be measured using the substrate BODIPY.
- Cells with high ALDH activity are often enriched in stem cell populations.
How can DNA replication be used for stem cell identification?
- Adult stem cells are often in a state of replicative quiescence.
- BrdU (bromodeoxyuridine) is used to track DNA replication.
- Cells incorporating BrdU can be visualized using fluorochrome-labeled antibodies.
What is lineage tracing in stem cell research?
Lineage tracing is a technique used to track cell ancestry and fate by marking a single cell and tracing its progeny over time.
What are the four important properties of tracers used in lineage tracing?
- Specificity – Marks only target cells.
- Stability – Remains detectable over time.
- Heritability – Passed to daughter cells.
- Non-toxicity – Does not interfere with cell function.
Why is quiescence important for stem cell identification?
Quiescent stem cells rarely divide, allowing long-term tracking via label-retaining techniques such as BrdU incorporation.
What is the significance of cutting-edge microscopy techniques in stem cell research?
Advanced microscopy enables precise localization of stem cells within tissues, improving our ability to study their niche and function.
What is the role of fluorescence-based approaches in stem cell research?
Fluorescence techniques detect cytoplasmic markers and metabolic features, supplementing other stem cell identification methods.
How can flow cytometry profiles distinguish between stem cells and differentiated cells?
Stem cells often exhibit a distinct dye-efflux pattern, such as the side population (SP) phenotype, which can be identified through fluorescence analysis.
Why is there no single strategy for identifying stem cells?
Stem cells have diverse characteristics, and no single method can reliably identify all stem cells. Instead, multiple approaches are used, including surface biomarkers, metabolic activity, and cell cycle analysis.
How do surface biomarkers help in stem cell identification?
Surface proteins specific to stem cells can be targeted using antibodies, allowing their isolation via techniques such as flow cytometry.
What are the two main types of stem cell surface protein names?
- Gene name – Describes function, origin, or structure.
- Designation – Assigned when the protein is recognized by a specific antibody.
What is flow cytometry used for in stem cell research?
Flow cytometry allows multi-parameter analysis of stem cells by detecting protein presence on the surface or inside cells.
What are the three main components of flow cytometry?
- Fluidics – Controls how cells pass through the laser.
- Optics – Uses lasers to detect fluorescence.
- Electronics – Processes signals from detected fluorescence.
What are ABC transporters, and how do they relate to stem cells?
ABC transporters are membrane proteins that pump out harmful substances. ABC-G2, highly expressed in stem cells, helps remove toxic dyes and chemicals.
How do fluorescence-based approaches help in stem cell detection?
Fluorescence dyes can be used to highlight cytoplasmic markers, allowing researchers to distinguish stem cells from differentiated cells.
What is the Hoechst dye efflux assay used for?
The Hoechst dye assay helps identify the side population (SP) phenotype, where cells that efficiently pump out the dye exhibit low fluorescence and are enriched in stem cell properties.
What is the SP phenotype, and how is it linked to stem cells?
SP cells actively expel Hoechst dye and appear with low fluorescence in flow cytometry. Many SP cells have strong stem-like properties.
What effect does Verapamil have on SP cells?
Verapamil blocks ABC transporters, reducing dye efflux and eliminating the SP phenotype, confirming the role of these proteins in stem cell activity.
How can metabolic activity be used to identify stem cells?
Stem cells often show distinct metabolic enzyme activity. High aldehyde dehydrogenase (ALDH) levels, measurable using BODIPY dye, are common in stem cells.
What is BrdU, and how is it used in stem cell identification?
BrdU (bromodeoxyuridine) is a thymine analog that integrates into newly synthesized DNA. Label-retaining stem cells that remain quiescent over time can be identified using BrdU-based fluorescence staining.
What is the significance of replicative quiescence in stem cells?
Many adult stem cells are quiescent, dividing infrequently to preserve their function. This property helps distinguish them from rapidly dividing progenitor cells.
What is lineage tracing, and why is it useful?
Lineage tracing is a technique that marks a single stem cell and tracks its progeny over time to study cell fate and differentiation pathways.
What are the four properties of good lineage tracers?
- Specificity – Marks only target cells.
- Stability – Remains detectable over time.
- Heritability – Passed to daughter cells.
- Non-toxicity – Does not affect cell function.
How do cutting-edge microscopy techniques aid stem cell research?
Advanced imaging methods allow precise localization of stem cells within their niche, helping researchers understand stem cell behavior and interactions.
How does fluorescence microscopy help detect stem cells?
Fluorescence microscopy can highlight cytoplasmic or nuclear markers in stem cells, enabling identification based on protein expression patterns.
Why do stem cells need to be identified using multiple techniques?
Stem cells share features with other cell types, so combining multiple methods (surface markers, metabolic activity, quiescence, lineage tracing) ensures accurate identification.
Magnetic Cell separation
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