lecture 24: cell separation techniques

Question 1

What is a reductionist approach to understanding cell biology?

Answer 1

• cell separative techniques allow scientists to study cells at a single population or single cell level
• single cell analysis can allow us to:
  
  • determine the relative abundance of stem cells within heterogeneous cell populations (i.e. disaggregated whole tissues)
  • examine gene and protein expression of individual cells, which avoids the mistake of taking the average of the entire cell population
  • discover the intrinsic and extrinsic signals that regulate the fate and specificity of stem cell populations
• stem cells are a minority population but have a big influence on structure, function and maintenance of tissues and organs

Question 2

What is the cellular diversity in the bone marrow?

Answer 2

• stem cells are very rare: less than 1 in 50,000 bone marrow cells
• stem and progenitor cells of differing potential are morphologically unrecognizable
• stem cells are defined operationally: by what they do

Question 3

What is lineage diversity in the lung?

Answer 3

• adult tissues contain multiple cell lineages and multiple stem cell populations

Question 4

What are the objectives of efficient cell separative techniques?

Answer 4

• resolution
  
  • how well are the cells of interest separated from the rest?
• enrichment
  
  • how homogeneous (pure) are the separated cells of interest?
• recovery
  
  • what proportion of the cells of interest that exist in the starting population are recovered in the enriched fraction?
• the outcome is always a compromise between purity and recovery

Question 5

What are physical methods of separating cells?

Answer 5

• physical methods exploit differences in physical properties of cells such as osmotic fragility, relative density and size
  
  • hypotonic lysis
  • density gradient centrifugation
• these are simple and powerful methods for resolving different cell types when used in appropriate settings
• each method has its advantages and limitations

Question 6

What is hypotonic lysis for red blood cell depletion?

Answer 6

• hypotonic lysis exploits the basic principles of osmotic pressure and the differential fragility of cells
• the basic cell structure of red blood cells makes them highly susceptible to hypotonic solutions
• isotonic solutions:
  
  • solute concentration inside cells is EQUAL to the solution outside the cells
  • amount of water transported into cells is equal to water transported out of cells
  • structural integrity of cells is maintained
• hypotonic solutions:
  
  • solute concentration inside cells is HIGHER than the solution outside the cells
  • water is transported into the cells
  • cells inflate and eventually burst

Question 7

What is density gradient separation of blood cells?

Answer 7

• density gradient centrifugation exploits the differences in cell density
• before centrifugation
  
  • whole blood
  • lymphoprop density gradient medium (density = 1.077g/mL)
• centrifuge 600 g, 30 min, 4 degrees C
• after centrifugation
  
  • plasma layer: platelets, cytokines, electrolytes
  • mononuclear cell layer: lymphocytes and monocytes
  • erythrocytes and polymorphonuclear cell layer: eoisonophils and neutrophils

Question 8

What is magnetic separation technology?

Answer 8

• combines the power of paramagnetic attraction and the specificty of antibodies to isolate cells from heterogeneous populations
• specific antibodies coupled to paramagnetic beads
• antibodies bind specifically to cells based on their immunophenotype
• bead-bound targets isolated by magnetic attraction
• surface activated dynabeads → add your ligand → ligand-coupled dynabeads → add sample containing target → dynabeads with bound target → magnetic separation → isolated, bead-bound targets are easily washed and concentrated in a final volume of your choice

Question 9

What is single cell separation by flow cytometry?

Answer 9

• flow cytometry is a laser based system that exploits the specificity of antibodies in conjuction with the precision of separating molecules on the basis of colour (emission of light)

Question 10

What are the advantages of flow cytometry?

Answer 10

• flow cytometry can be defined as the simultaneous measurement of multiple physical characteristics of a single cell as the cell flows in suspension through a laser detection system
• high resolution
  
  • identifies multiple parameters for every single cell
  • dependent on specificity of antibodies, but simultaneous acquisition of information about multiple properties of a single cell in real time increases specifity
• high enrichment
  
  • cells can be sorted as single cells
• high recovery
  
  • can sort up to 50,000 cells/sec
  • can sort through large samples relatively quickly

Question 11

What are the basic principles of flow cytometry?

Answer 11

• heterogeneous lung cells stained with specific markers
• flow cytometer sorts individual cells
• sorter deflect cells of interest into collection tubes
• sorted cell for functional analysis
• electronic gating of cells

Question 12

What is the flow cytometer machine?

Answer 12

• BD FACS Aria III - Melbourne Brain Centre, University of Melbourne

Question 13

What are functional components of the flow cytometer?

Answer 13

• a flow cytometer is comprised of the following specialised components:
  
  • fluidics:
    
    • the fluidics system focuses individual cells in the stream (sheath fluid) so that they pass through the laser focus point as single events
  • optics:
    
    • a laser beam is used to excite a fluorescent tag (e.g. a fluorescent antibody) bound to the cells in the sample
  • electronics:
    
    • photodiode detectors or photomultiplier tubes collect the light emitted from fluorescent tags bound to the cells and convert this into a pulse of electrical current
    • this intensity of this pulse is converted into a digital signal which allows us to quantify the amount of fluorescent tag bound to individual cells

Question 14

What is fluidics in flow cytometry?

Answer 14

• focusing in on single cells
• sample is injected into a steady stream of sheath fluid under positive pressure
• this results in hydrodynamic focusing which forces the cells to pass through the laser optics as single events

Question 15

What are light scatter characteristics of cells?

Answer 15

• optics
• side scatter provides an indication of cellular complexity
• forward scatter provides an indication of relative cell size

Question 16

What is spectral separation of fluorescent tags?

Answer 16

• the flexibility of flow cytometry comes from the ability to accurately detect the emission of light (colour) of different fluorescent tags
• different fluorescent molecules have different excitation and emission spectra
• dichroic filters allow a narrow band of colours (wavelengths) to pass through while reflecting others
• bandpass filters allow a narrow band of colours (wavelengths) to pass through while rejecting others
• photomultiplier tubes convert fluorescence emission into a quantifiable electrical signal
• multiple detectors for single lasers increases the number of parameters that can be detected
• multiple lasers further increase the number of parameters that can be detected
• cells pass sequentially through different lasers, which allows colours with overlapping emission spectra but different excitation spectra to be collected for the same sample

Question 17

What are characteristics of different fluorescent tags?

Answer 17

• conjugation of fluorescent tags to different antibodies or molecules allows multiple parameters for every single cell

Question 18

What are parameters that can be measured by flow cytometry?

Answer 18

• there is an ever growing repertoire of cellular and molecular reagents and probes which can be used to interrogate cell phenotype and function
  
  • cell surface antigens
  • intracellular antigens
  • quantum dots
  • membrane integrity, and turnover, viability
  • DNA and RNA content
  • protein content
  • cell surface sugars
  • RNA content
  • cell lipids
  • receptors
  • enzyme activity
  • membrane potential and cellular ionic environment
  • intracellular calcium
• note: some parameters require fixation of cells or are toxic to cells and can not be used to isolate viable cells

Question 19

What is data acquisition?

Answer 19

• photomultiplier tubes convert colour intensity into an electronic signal
• intensity is converted to quantifiable digital signal that can be expressed in different
• for dotplots, every dot on the screen represents a single cell
• electronic gating tells the sorter what to sort

Question 20

What is collection of cells of interest?

Answer 20

• the sort chamber
• nozzle
• deflection plates:
  
  • cells of interest are deflected into collection tubes for subsequent analysis (i.e. cell culture etc)
• collection tubes

Question 21

What is spectral overlap?

Answer 21

• the way the detectors work is they usually have a filter that allows a certain amount of light to come through
• this might be 530nm → some light might get though that is not this e.g. 545
• leakage of R-PE fluorescence detected as Alexa-Fluor 488 false-positive signal, leakage of alex fluor 488 fluorescence detected as R-PE false-positive signal
• machine allows you to compensate for this
• can’t compensate for this in microscopy but can in FACS

Question 22

What are limitations and caveats of flow cytometry?

Answer 22

• cell preparation
  
  • must have single cell suspensions
• the separation is only as good as the reagents
  
  • purity, specificity and signal-to-noise ratio
• incidence of the target cell
  
  • if incidence of target is 1:50,000; then 10 hours sorting at 20,000 events per second to yielf 100,000 purified cells
  • requirement for combining pre-enrichment methods with high recovery with potentially low resolution and/or enrichment
• what you see is not necessary an accurate description of the population
  
  • need for appropriate controls and fluorescence compensation

Question 23

What are Shapiro’s laws of flow cytometry?

Answer 23

• 0th law: there is no magic
• 1st law: A 51 µm clogs a 50µm orifice
• 2nd law: what you see is what you get
• 6th law: there are some cell identification problems that even monoclonal antibodies can’t solve
• 7th law: no method of analysis can make good data out bad data
• 8th law: know your cells, know your instrument