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Molecular Basis Of Disease > Flow Cytometry > Flashcards

Flow Cytometry Flashcards

1
Q

What is flow cytometry?

A

A technique which simultaneously measures several physical characteristics belonging to a single cell in suspension.

OR

Measuring properties of cells in flow

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2
Q

What is flow sorting?

A

Sorting cells based on properties measured in flow

Allows us to isolate very rare cells so we can do further analysis on them

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3
Q

What can a flow cytometer tell us about a cell?

A
  1. It’s relative size
  2. It’s relative granularity/internal complexity
  3. It’s relative fluorescence intensity
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4
Q

What are the two methods of visualisation?

A

Fluorescence microscopy

Flow cytometry - can be used to quantify rare cells and more accurately

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5
Q

What are the 3 stages of flow cytometry?

A

Fluidics

Optics

Electronics

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6
Q

What occurs during the fluidics stage of flow cytometry?

A

Cells in suspension

Flow in single file through a illuminated volume

Accomplished by injecting sample into sheath fluid as it passes through a smooth office

When sample fluid flows in a central core that does not mix with sheath fluid - laminar flow

The introduction of a large volume to a small volume - hydrodynamic focusing

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7
Q

What occurs in the optics part of flow cytometry?

A

Laser fired at cells
The cells scatter light and emit fluorescence that is collected and filtered and converted to digital values

Lasers:
- single wavelengths of light
- can be inexpensive, air cooled units, or expensive water cooled units
- provide coherent light

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8
Q

What occurs in electronics stage of flow cytometry?

A

Digital values stored on a computer

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9
Q

Label a simple diagram of a flow cytometer

A

Label a simple diagram of the channel layout in a flow cytometer

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10
Q

What is stroke shift?

A

The energy difference between the lowest energy peak of absorbance and the highest energy of emission.

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11
Q

Give examples of the fluorochromes and they’re respective dyes

A

Fluorescein isothiocyanate (FITC) - Green

Phycoerythrin. (PE) - Orange

Peridinin chlorophyll protein (PerCP) - Red

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12
Q

What is fluorescence?

A

When laser hits a fluorochrome and it’s excited at one wave length and then when it goes back to its unexpired state it emits fluorescence at a higher wavelength

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13
Q

Explain why combinations of fluorochromes can be used together?

A

Excited by the same laser but emit at different wave lengths

The filters and mirrors help to reduce overlap of light so we can analyse data from multiple fluorochromes together.

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14
Q

What are ideal samples to use for flow cytometry?

A

Single cells in suspension

  • bone marrow
  • peripheral blood
  • fine needs aspirate
  • CSF
  • fresh tissue
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15
Q

What are the two ways of labelling cell surface proteins?

A

Direct: monoclonal antibodies (MoAbs) are pre-conjugated to fluorochromes
-uses one florescently labelled antibody to label cell

Indirect: un-cojugated MoAbs
- uses two antibody’s, with one being florescently labelled to label cell

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16
Q

What are the two most common ways of representing the data of flow cytometry?

A

Histogram
-one dimensional display

Dot plot
- two dimensional (can analyse 2 parameters at the same time)

17
Q

What is gating?

A

Using our computer we can draw a gate over a population that we want to look at further on the basis of they’re florescence

  • can further analyse by only showing cells in a certain parameter
18
Q

How is flow photometry data analysed?

A
19
Q

What is the simplest univariate cell cycle method?

A

Cellular DNA is detected using a fluorescent dye that binds preferably to DNA

Protium iodide is most commonly used

But it required permiabilization of the plasma membrane

20
Q

Describe how a PI assay works

A

PI cannot normally cross 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

21
Q

What are some characteristic of apoptosis?

A

Condensation of chromatin

Blabbing of nuclear material

Internucleosomal degradation of DNA

22
Q

What are 3 detection methods for apoptosis?

A

By staining with the PI

Phosphatidyl serine, can be detected by incubating the cells with fluorescent labeled annexin V and PI

By staining with 7-aminoactinmycin D

23
Q

What is 7-AAD?

A

Excitation: 488nm

Emission: 660nm

DNA-specific

Long emission wavelength

24
Q

What are the applications of flow cytometry?

A
  • immunophenotyping of leukaemia and lumphomas
  • detection of MRD
  • stem cell enumeration
  • CD4/CD8 in HIV
  • measurement of intracellular cytokines
  • measurement of cell proliferation
  • study of cell cycle and apoptosis
    - asses,ent of transfection efficiency

Molecular Basis Of Disease (32 decks)

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