10 Questions
What is flow cytometry used for?
analysis of multiple parameters of individual cells within heterogeneous populations
Flow cytometry performs analysis by passing thousands of cells per second through a __________ beam.
laser
What does the data gathered by a flow cytometer allow for the analysis of?
Complexity and health
Side scatter in flow cytometry is caused by the size of the cell.
False
Match the biological cell populations with their descriptions:
Lymphocytes = Small cells possessing low internal complexity Monocytes = Medium-sized cells with slightly more internal complexity Neutrophils and other granulocytes = Large cells that have a lot of internal complexity
What is the purpose of using the cell permeant reagent 2’,7’ –dichlorofluorescein diacetate (DCFDA) in flow cytometry?
Measure hydroxyl, peroxyl, and other reactive oxygen species (ROS) activity within the cell.
Which dye is commonly used for staining DNA in flow cytometry?
Propidium Iodide (PI)
Define ploidy in the context of flow cytometry.
Ploidy refers to the number of chromosomes in a cell.
Is the translocation of phosphatidyl serine (PS) to the outer leaflet of the plasma membrane a characteristic of apoptotic cells?
True
What are the potential consequences of mitochondrial dysfunction resulting from disease or compound treatment?
Cell death by apoptosis or cellular energetic decline.
Study Notes
Flow Cytometry
- Flow cytometry is a powerful technique for analyzing multiple parameters of individual cells within heterogeneous populations.
- It involves passing thousands of cells per second through a laser beam and capturing the light that emerges from each cell.
Instrumentation
- Fluidic system: presents samples to the interrogation point and takes away waste.
- Lasers: the light source for scatter and fluorescence.
- Optics: gather and direct the light.
- Detectors: receive the light and convert signals into digital data.
- Electronics and peripheral computer system: perform the necessary analyses.
Forward Scatter Histogram
- Measures cell size: magnitude of voltage pulse recorded for each cell is proportional to the cell size.
- A histogram of forward-scatter data is a graphical representation of the size distribution within the population.
Side Scatter Histogram
- Measures cell complexity: side scatter is caused by granularity and structural complexity inside the cell.
- Signals collected by the side-scatter detector can be plotted on one-dimensional histograms.
- Two-dimensional dot or scatter plots can be used to correlate size and complexity of cell populations.
Fluorescence
- Involves the use of fluorescent molecules such as fluorophore-labeled antibodies to analyze cellular characteristics.
- Fluorescent signal is detected by the flow cytometer and translated into a voltage pulse proportional to the amount of fluorescence emitted.
Data Analysis
- Linear or log scale histograms can be used to graphically represent the data.
- Two-parameter dot plots can be used to look at two parameters at once.
- Gating: restricting the analysis to a specific cell population by drawing a region around it.
- Compensation: correcting for spectral overlap between fluorophores to accurately record the fluorescence signal.
Applications of Flow Cytometry
- Cell signaling: monitoring intracellular checkpoints simultaneously to analyze complex cell events.
- Cell health and quality: measuring mitochondrial superoxide generation and membrane integrity to assess cell health.
- Ploidy analysis: measuring DNA content to obtain information about ploidy, relevant in tumor cell characterization and cell cycle analysis.
This quiz covers the application of flow cytometry, chromatography, and mass spectroscopy in clinical diagnosis and basic research. It is a part of the instrumentation in medical diagnostic, laboratory, and blood banking course.
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