week 2: flow cytometry & enzyme assays Flashcards
section 2 & 3 (52 cards)
What is Flow Cytometry?
A technique to measure multiple physical characteristics (size, granularity, fluorescence) of individual cells flowing in a suspension.
What is the rate of cell analysis in Flow Cytometry?
Typically 500–20,000 cells per second.
Main components of a Flow Cytometer?
Fluidics (delivers single cells to laser)
Optics (excitation source, flow cell, detectors)
Electronics (signal conversion and data analysis)
What maintains cells in a straight line through the laser beam?
Hydrodynamic focusing.
What are common lasers used in Flow Cytometry, and why?
Diode lasers (e.g., 488 nm) because they are cost-effective, compact, and efficient at exciting common fluorochromes (e.g., FITC, PE).
Role of lasers in Flow Cytometry?
Lasers produce monochromatic, coherent light that excites fluorescently labelled cells, causing emission of fluorescence detectable by sensors.
What is fluorescence emission in Flow Cytometry?
A fluorochrome absorbs light at one wavelength (excitation), moves to a higher-energy state, then returns to a lower-energy state, emitting light of a longer wavelength (emission).
Common fluorochromes used?
FITC (fluorescein isothiocyanate), PE (phycoerythrin).
Why use multiple fluorochromes in Flow Cytometry?
Allows simultaneous detection of different cell markers.
What does Forward Scatter (FSC) measure?
Measures cell size; larger cells scatter more light forward.
What does Side Scatter (SSC) measure?
Indicates internal complexity or granularity; higher SSC suggests more internal structure.
How is Flow Cytometry data typically displayed?
Histograms: display single parameter (intensity)
Dot plots: show two parameters simultaneously (e.g., FSC vs SSC)
Define gating in Flow Cytometry.
Selecting a subset of cells for detailed analysis based on specific criteria.
Why use Multi-Colour Flow Cytometry?
Identifies multiple cell markers simultaneously, saves time, resources, and allows smaller sample volumes.
What’s special about Luminex Technology?
Measures multiple analytes simultaneously (up to 100) using bead-based assays.
Difference between Flow Cytometry and Cell Sorting?
Cell Sorting physically separates specific cell populations identified by Flow Cytometry.
Methods of cell sorting?
Electrostatic deflection or mechanical sorting.
What purity can cell sorting achieve?
Typically greater than 95%.
Common samples analysed in Flow Cytometry?
Blood cells, cultured cells, processed tissues, bone marrow.
How do droplet formation issues affect sorting?
They may lead to incorrect charging of droplets and lower sorting purity.
What are photomultiplier tubes (PMTs)?
Sensitive detectors in Flow Cytometry used for amplifying weak fluorescent signals.
What does “compensation” in Flow Cytometry mean?
Correcting spectral overlap between fluorochrome emissions.
What unique advantages does Flow Cytometry offer?
High throughput (500–20,000 cells/sec)
Single-cell resolution
Ability to identify rare cell populations (e.g., <0.0001%)
Multiparametric analysis (size, granularity, fluorescence)
What is hydrodynamic focusing?
A process using sheath fluid (isotonic saline or PBS) to position cells in single file at the centre of a fluid stream, enabling precise analysis by lasers.
