Lecture 17: Flow Cytometry

Question 1

overview of flow cytometry

Answer 1

• The measurement of cells/beads/similar in flow – delivers them singly past a point of measurement.
• Instruments use light focused at the point of measurement and then scattered light and fluorescence of different wavelengths are recorded.
• Typically, light scatter at two different angles and from one or more fluorescences will be measured.
• Usually, cells being analysed but other particles, like nuclei, chromosomes, or small beads can also be studied

Question 2

pros

Answer 2

• Measurements on large numbers of single cells within a short period of time
• Reveal heterogeneity; identify and quantify different subsets of cells
• Physically sort cells of interest for further study
• Suitable for clinical applications (blood)

Question 3

cons

Answer 3

• Requires suspension of single cells or other particles, with minimum clumps and debris
• Information about tissue architecture and the spatial relationship between different cells is lost during sample prep

Question 4

uses

Answer 4

• Analysing expression of cell surface and intracellular molecules
• Characterising and defining different cell types in heterogeneous cell population (e.g. blood)
• Assessing purity of isolated cell subtypes
• Analysing cell size and volume

Question 5

an EVENT

Answer 5

• Info from a single cell or particle
• Each event is given a channel number based on its measured intensity
• The more intense the fluorescence the higher the assigned channel number (up to 1024)

Question 6

forward scatter

Answer 6

• Forward scatter (FSC) – correlates with cell size

Question 7

side scatter

Answer 7

• Side scatter (SSC) – correlates with complexity/granularity as scattering depends on density of cells/particles

Question 8

Fluorochromes/fluorophores:

Answer 8

• Non-covalently bound to structures within cells, e.g., propidium iodide (PI) is a DNA stain that is weakly fluorescent in water but fluorescence increases 50-fold on intercalating with DNA
• Covalently bound to probes such as antibodies, e.g., fluorescein isothiocyanate (FITC) reacts with amino group on lysine residues
• Reporter molecules used to show a gene construct has entered a cell, e.g., GFP – green fluorescent protein
• CyTOF Mass Cytometry – metal-conjugated antibodies
• Quantum dots - fluorescent nanocrystals of 2-20nm diameter with wide absorption spectrum

Question 9

Fluorescence:

Answer 9

• Absorb light energy and electrons raised from ground state to excited state
• Electron returns to ground state by a variety of transitions which may involve the emission of a quantum of light – fluorescence
• Emitted light is of lower energy and longer wavelength than the exciting light
• Emitted light can be separated using filters

Question 10

Fluidics

Answer 10

• Sheath fluid driven trhough flow chamber by pressure
• Pressure also forces samples into the sheath fluid
• Pressure regulator to control rate of sample flow
• Generally 3 fixed settings – low, medium and high
• Pinch value or syringe pump controls sample delivery
• Vacuum system clears the flow cell and enables cleaning

Question 11

optics

Answer 11

• Light sources
• Typically lasers
• High intensity beam of monochromatic light
• Small spot size = focus onto small volume
  o Max excitation of cell
  o Increased likelihood of once cell at a time
• Lasers are typically air-cooled solid state
  o Argon ion producing blue at 488nm
  o Typically a He-Ne at 633nm
  o Varying wavelengths; 355nm, 405nm, 550nm, and 780nm

Question 12

Measuring light fluorescence:

Answer 12

• Light from cell/particle is split into defined wavelengths
• Light channelled via filters and mirrors
• Filters ensure that sensors detect specific wavelength
• Sensors are called photomultiplier tubes (PMTs)

Question 13

Applications:

Answer 13

• Clinical use
• Cell viability
• Cell analysis
• Intracellular flow cytometry
  o Monitor changes in phosphorylation (alternative to immunoblotting)
• Cell proliferation

Question 14

CyTOF – mass cytometry:

Answer 14

• Cells stained with a panel of rare element metal-conjugated probes (natural concentration in biological samples is below the detection limit)
• Cells individually atomised and ionised
• Cloud of atomic ions for each single cell extracted into the ion optics/time-of-flight regions for separation by mass
• Intensity of the signal is directly proportional to the number of specific probe-derived ions striking the detector and thus the number of antibodies originally bound per cell