Flow Cytometry - Introduction and applications

Question 1

Explain what is flow cytometry

Answer 1

• Measuring properties of cells in flow
• Sorting (separating) cells based on properties measured in flow
• Also called Fluorescence-Activated Cell Sorting (FACS)

Question 2

What can flow cytometry tell us about cells

Answer 2

• Its Relative Size
• Its Relative Granularity/Internal Complexity
• Its Relative Fluorescence Intensity

Question 3

List the 2 methods of visualisation

Answer 3

• Fluorescence Microscopy
• Flow Cytometry

Question 4

What are the components involved in flow cytometry

Answer 4

• Light source
• Flow chamber
• Optical system
• Light detectors
• Computers

Question 5

Explain the fluidics portion of the system

Answer 5

• Need to have cells in suspension flow in single file
• Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
• Sample fluid flows in a central core that does not mix with the sheath fluid - Laminar flow
• Introduction of a large volume into a small volume - Hydrodynamic Focusing

Question 6

What does each of the laser scatters show

Answer 6

• Forward light scatter proportional to size
• 90° Light scatter proportional to granularity

Question 7

Explain channel layout for laser-based flow cytometry

Answer 7

• laser light is filtered by dichroic filters to block other wavelengths
• 3 different colours are used to look for 3 different antibodies

Question 8

how are the signals from detectors processed

Answer 8

• analog-digital conversion of signals

Question 9

Define fluorescence and what is stokes shift

Answer 9

• fluorescence is the emission of energy at a different wavelength than what it absorbed
• The energy difference between the lowest energy peak of absorbence and the highest energy of emission

Question 10

List some fluorochromes and what colour they emit

Answer 10

Fluorescein isothiocyanate (FITC) - GREEN
Phycoerthrin (PE) - ORANGE
Peridinin Chlorophyll Protein (PerCP) - RED

Question 11

What are the methods of labelling

Answer 11

DIRECT - Monoclonal antibodies (MoAbs) conjugated to fluorochromes

INDIRECT - Unconjugated MoAbs and secondary antibodies conjugated to fluorochromes

Question 12

How can data from flow cytometry be displayed

Answer 12

• Histograms
• Dot Plot

Question 13

Explain how we can analyse data from flow cytometry

Answer 13

• Select a population of interest and apply new analysis parameters to it
• can be done to either box plots or histograms

Question 14

What are cell cycle methods

Answer 14

• In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
• Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization of the plasma membrane

Question 15

How do Propidium iodide assays work

Answer 15

• PI cannot normally cross the cell membrane
• If the PI penetrates the cell membrane, it is assumed to be damaged
• Cells that are brightly fluorescent with the PI are damaged or dead

Question 16

how could we measure apoptosis?

Answer 16

• Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”
• Characteristics are condensation of the chromatin material
• Blebbing of nuclear material
• Often accompanied by internucleosomal degradation of DNA giving rise to distinctive ‘ladder’ pattern on DNA gel electrophoresis

Question 17

What are the methods for detecting apoptosis

Answer 17

• By staining with the dye PI (cells fixed)
• Phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)
• By staining with 7-aminoactinomycin D (cells not fixed)

Question 18

How does 7-aminoactinomycin (7-AAD)

Answer 18

Ex: ~488 nm

Em: ~660 nm

DNA-specific:
intercalates in G-C regions

long emission wavelength:
with FITC & PE labelled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex

Question 19

Explain how we can sort the samples as they are being examined

Answer 19

The machine vibrates and separates cells by charging them and deflecting them into tubes. the sorting is based on parameters set by the user.

Question 20

What are the applications of using flow cytometery

Answer 20

• Immunophenotyping of leukaemias & lymphomas
• Detection of MRD
• Stem cell enumeration
• CD4/CD8 in HIV
• Measurement of intracellular cytokines
• Study of cell cycle, viability & apoptosis
• Measurement of cell proliferation
• Assessment of transfection efficiency