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TI > Flow Cytometry > Flashcards

Flow Cytometry Flashcards

1
Q

Briefly define flow cytometry

A
  • measures properties of a cell in flow

- sorts cells based on properties measured - Fluoresence-activated cell sorting (FACS)

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2
Q

What 3 things can flow cytometry can tell us about a cell?

A
  • its relative size
  • its relative granularity/internal complexity
  • its relative fluorescence intensity
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3
Q

What are the main differences between flow cytometry and fluorescence microscopy?

A
  • Fluorescence microscopy you’d label the surface antigen and look down a microscope, then have to scan through hundreds of fields to count the number of antigens on each cell
  • with FC, you can get results from thousands of cells simultaneously - very quantitative
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4
Q

What are the 3 main stages of flow cytometry?

A
  • Fluidics - cells in suspension flow in single file through an illuminated volume
  • Optics - where they scatter light and emit fluorescence, and its collected and filtered
  • Electronics - It is converted to digital values and stored on a computer
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5
Q

What are the major components of a flow cytometer?

A
  • light source
  • flow chamber
  • optical system
  • light detector
  • computer
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6
Q

How do you get cells to flow down the nozzle in single file?

A
  • need to have cells in suspension
  • inject the sample into sheath fluid and pass it through a narrow tip
  • sample fluid flows in a central core that doesn’t mix with sheath fluid
  • introduction of a large volume into a small volume = hydrodynamic focusing
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7
Q

What are the two sorts of light scatter we can measure?

A
  • Forward scatter - light that hits the sensor in a forward direction is proportional to the size
  • Side scatter - light that hits and goes of a right angle is proportional to granularity
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8
Q

How can we measure the different wavelengths of light emitted separately?

A
  • Light emitted passes through different filters to photomultiplying tubes
  • The PMTs detect a very narrow wavelength
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9
Q

What is Stoke’s shift?

A
  • The energy difference between the lowest energy peak of absorbance and the highest energy of emission
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10
Q

What is a fluorochrome?

A
  • A fluorescent tag

- When it is excited by the laser, it emits light at a longer wavelength

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11
Q

Give the name of 3 commonly used fluorochromes and the colour light they emit

A
  • FITC = green
  • PE = Orange
  • PerCP = Red
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12
Q

What are common sources of cells for flow cytometry?

A
  • peripheral blood
  • bone marrow
  • fine needle aspirate
  • fresh tissue (need to prepare in a suspension)
  • CSF and other fluids
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13
Q

What is the direct method of labelling?

A
  • use preconjucated monoclonal antibodies
  • (flurochrome is already attached)
  • e.g. if we want to quantitate the number of CD3 antigens, we use an anti-CD3 pre-conjugated with the fluorochrome
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14
Q

What is the indirect method of labelling?

A
  • 2 step procedure

- e.g. use an anti-CD3 Ab, then a non-specific one that is conjugated to a fluorochrome

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15
Q

What are the 2 common ways of displaying the data?

A

Histogram or dot plot

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16
Q

What can we see from a histogram?

A
  • a cell count on the y axis, and fluorescence intensity on the x axis
  • gives the number of cells at a give intensity
17
Q

What can we see from a dot plot?

A
  • you can display any 2 parameters you want

- e.g. side scatter (granularity) and size (forward scatter)

18
Q

What is gating?

A
  • you can select a region on a graph that you want to investigate further - e.g. just lymphocytes
  • the computer will colour these however you want, then make a dot plot of just lymphocytes in this region.
19
Q

What is the advantage of using more than one fluorochrome at once?

A
  • can quantitate lots of populations at once
  • can measure many different parameters at once
  • e.g. with 3, you can look at 8 populations
20
Q

What molecule is commonly used to analyse cell cycle distributions?

A

Propidium Iodide - undergoes a dramatic increase in fluorescence upon binding DNA

21
Q

How can we measure cell viability with PI?

A
  • PI cannot normally cross the cell membrane
  • if the PI penetrates the membrane, it is assumed to be damaged
  • brightly fluorescing cells are damaged or dead
  • The viable cell will appear negative as the PI cannot get in
22
Q

Apoptosis vs Necrosis

A
  • Apoptosis = programmed cell death, apoptotic bodies form with functioning organelles
  • Necrosis = cell ruptures and releases its contents, non-functioning organelles
23
Q

What does the sub-G0 peak mean?

A
  • if cells are apoptosis, when viewing cell cycle on a histogram, there will be a peak before G0
24
Q

How can we detect apoptosis?

A
  • Phosphatidyl serine can be detected by incubating cells with Annexin-V (bound to FITC) and PI.
  • PS is usually expressed on the inside of plasma membranes
  • Therefore live cells wont bind the annexin as PS is inside and the PI won’t be able to get inside
  • In early apoptosis, PS flips to the outside, so annexin can bind, however the PI can still not get in
  • In late apoptosis, the membrane ruptures and so both can bind
  • 7AAD can also bind to DNA in late apoptosis = red
  • Allows all 3 stages to be quantitated
25
Q

Give 5 applications of FC

A
  • Immunophenotyping of leukaemias and lymphomas - diagnose stages with haematological neoplasm
  • detection of minimal residual disease - neoplastic cell concentration is lower than 5%
  • stem cell enumeration - for bone marrow transplants, count levels of CD34 in peripheral blood to see if enough bone marrow has been released following GH injections
  • Organ transplant - check that there aren’t antibodies in donor or patient serum which could cause rejection
  • Cd4/8 in HIV - clinically stage disease progression, measure no. of CD4+ cells because they get destroyed in HIV patient, the more CD4+ cells, the less severe case
  • measurement of IC cytokines
  • study of cell cycle, viability and apoptosis
  • measurement of cell proliferation
  • assessment of transfection efficiency
26
Q

What is the difference between a cell sorting machine and flow cytometer?

A
  • nozzle is vibrated - flow in a stream but when a charge is added, cells break off.
  • these now charged cells are then sorted into positive and negative
  • We can administer a charge when the cell we want is right at the end of the nozzle
  • can sort two parameters at once using the different charges
27
Q

What is 7AAD?

A
  • excited by 488nm laser - emits red light at 660nm
  • DNA specific - binds to G-C regions
  • Used to detect apoptosis and capase activity

TI (18 decks)

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