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Flashcards in Flow Cytometry I + II Deck (29)
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Define flow cytometry

Measures properties in flow of single cells in suspension

Carried out using light scatter and fluorescence


Define flow sorting

Sorting (separating) cells based on properties in flow Also called FACS (fluorescence-activated cell sorting)


What can a flow cytometer tell us about a cell?

1. Its relative size

2. Its relative granularity/ Internal complexity

3. Its relative fluorescence intensity


What can the relative fluorescent intensity be used for? 

Can be used to look at different characteristics of the cell such as: 

  • Cell surface receptors 
  • Adhesion molecules 
  • Levels of intracellular cytokines and enzymes 
  • DNA 


What are some of the ways in which we are able to visuallise fluorescent cells? 

  • Fluorescence microscopy 
    • Not very quantitative as it takes a long time to accurately quantitate the cells 
  • Flow cytometry 
    • Looking at thousands of cells, able to asses the intensity of each individual cell


Summarise the steps of a flow cytometer

Fluidics → Where cells in suspension flow single file 

Optics → The cells flow through an illuminated volume where they scatter light and emit fluorescence 

Electronics → The fluorescence is collected, filtered and converted into digital values which are stored on the computer 


Describe fluidics in flow cytometry

  • Cells will be introduced and forced out single file in suspension flow 
  • This is accomplished by injecting a sample into a sheath fluid as it passes through a small orifice 
  • A laser will hit the flow cells and light will be scattered due to cell size and granularity 
  • Or cells have been stained with fluorescent markers, the cells will also emit fluorescence 


Define laminar flow and hydrodynamic focusing in terms of glow cytometry

Laminar flow = sample fluid flows in a central core that doesnt fix with sheath fluid

Hydrodynamic focusing = Introduction of a large volume into a small volume


Describe the optics part of the flow cytometer

Focus on laser 

  • It is a single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths 
    • It can provide from milliwatts to watts of light 
    • Can be inexpensive, air-cooled units/ expensive, water cooled units 
      • They provide coherant light (single frequency) 


What happens when the laser hits a cell?  

When the laser hits the cell light will be scattered in two directions 

1) Light is scattered in a forward direction which is proportional to the size of the cell 

2) Light will be scattered in a 90 degree angle which is proportional to the granularity of the cell 


Describe the electronics part of flow cytometry 

It is where light signals from detectors is converted into a digital signal 



Describe the basis of fluorescence 

When a fluorochrome is excited by a laser at a certain wavelength the light it emits following resumption back to its unexcited state is at a longer wavelength 

There are two wavelengths 

  1. Emission excitation wavelength 
  2. Emission wavelength 


What is Stokes Shift

Is the energy difference between the lowest energy peak of absorbance and the highest energy peak of emission 


What is a fluorochrome and give examples of some common fluorochromes

A fluorochrome is a chemical which can absorb energy from an excitation source (laser beam) and emit photons at a longer wavelength (fluorescence), which is captured by optical detectors of the flow cytometer

Fluorescein isothiocyanate (FITC)     GREEN

Phycoerythrin (PE)                                   ORANGE

Peridinin Chlorophyll Protein (PerCP)   RED


Give some examples of single cells in suspension

  • Peripheral blood 
  • Bone marrow 
  • Fine needle aspirate 
  • CSF and other fluids
  • Fresh tissue 


What are the two methods of labelling in immunofluorescence

1) Direct - monoclonal antibodies are preconjugated to fluorochromes 

2) Indirect - unconjugated MoAbs are added (primary, then secondary) and fluorophores are added on top of this 



What are advantages and disadvanatages of indirect antigen labelling? 

+ Gives an amplified signal 

- Get more background staining 


How is data following a flow cytometer presented? 

Histogram = Where you can only measure 1 parameter at a time 

Dot Plot = able to quantitate on the basis of two parameters (e.g forward scatter and side scatter) 


What was one of the early applications of flow cytometry? 

One of the early applications of flow cytometry was the analysis of cell cycle position by quantitation of cellular DNA 

Flow cytometry is still the method of choice for fast and accurate determination of cell cyle distributions 


How is cellular DNA detected in flow cytometry (for example analysis of the cell cycle> 

Cellular DNA is detected using fluorescent dye which preferentially binds to DNA 

Propidium iodide is the most commonly used dye 

  • It undergoes a dramatic increase in fluorescence upon binding DNA.
  • It requires permeabilization of the plasma membrane which occurs in late apoptosis (must be late apoptosis or dead cells)


Explain how propidium iodide works

  • Normally propidium iodide cannot cross the membrane 
  • If propidium iodide penetrates the membrane it is assumed to be damaged 
  • Cells that are brightly fluorescent with PI are severly damaged or dead 


What is apoptosis and describe some characteristics

Apoptosis = Programmed cell death where cell goes through a regulated process of dying 

Characteristics are: 

  • Condensation of chromatin material 
  • Blebbing of nuclear materal 
  • Internucleosomal degradation of DNA giving rise to ladder pattern on DNA gel electrophoresis 


What is the difference between apoptosis compared to necrosis? 


What detection methods can be used for apoptosis? 

  • Staining dye with propidium iodide 
  • Phosphatidyl serine can be detected by incubating cells with fluorescein-labeled Annexin and propidium iodide 
  • Staning with 7-aminoactinomycin D


How can you distinguish different populations of cells using propidium iodide and annexin? 

 ⇒ If the cell doesnt show active AnnexinV or PI - Live cell 

 ⇒ If the cell shows AnnexinV but not PI - Early apoptotic cell 

 ⇒ If the cell shows both AnnexinV and PI - Late apoptotic/necrotic cell


Describe 7-Aminoactinomycin D (7-AAD)

  • DNA-specific (intercalates in G-C regions) 
  • Long emission wavelength 
    • We can use this dye in conjunction with other antibodies and fluorochromes 
      • Allows us to carry out FITC and PE labelled antibodies alongside for simulatenous evaluation of DNA content and 2 colour immunofluoresence using a 488nm common laser


What are applications of flow cytometry? 

  • Immunophenotyping of leukaemia’s & lymphomas
  • Detection of MRD (minimal residual disease)
  • Stem cell enumeration (separating, because of how rare stem cells are)
  • CD4/CD8 in HIV
    • Detects the proportion of CD4 and CD8
  • Measurement of intracellular cytokines
  • Study of cell cycle, viability & apoptosis
  • Measurement of cell proliferation
  • Assessment of transfection efficiency
    • Using GFP


Describe cell sorting 

  • Still follows the same principle as cytometry - the cells are in suspension single file, a laser is put on them and light is picked up and emitted telling a computer what type of cell it is 
  • In cell sorting we can draw a range around the type of cells we want so the computer will sort them out 
    • The nozzle tip will always be vibrating so that the stream will break into droplets 
    • If this droplet contains the desired cell a charge will be added to cells of choice 
    • The charged droplets containing the cell will be collected into tubes by being pulled towards a deflection plate 
    • The rest of the cells will be collected for waste 

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