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Molecular Basis Of Disease/ Microbiology And Immunology > Flow Cytometry- Introduction And Applications > Flashcards

Flow Cytometry- Introduction And Applications Flashcards

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Biology 101 flashcards
1
Q

What is flow cytometry?

A

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

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2
Q

What is the definition of flow cytometry?

A

Measuring the properties of cells in flow

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3
Q

What is flow sorting?

A

Sorting (separating) cells based on properties measured in flow

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4
Q

What is another name for flow sorting?

A

Fluorescence-activated cell sorting (FACS)

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5
Q

What can a flow cytometer tell us about a cell?

A

Relative size,
relative granularity/ internal complexity,
Relative fluorescence intensity

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6
Q

What can we measure with a flow cytometer?

A

Surface receptors
Intracellular cytokines/enzymes
Measure cell cycle viability and apoptosis

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7
Q

What are fluidics?

A

Cells in suspension flowing in a single file

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8
Q

How do fluidics work?

A

Inject a sample into a sheath fluid as it passes through a small hole
Simple fluid flows down a central core

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9
Q

What is hydrodynamic focusing?

A

Introduction of a large volume into a smaller volume

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10
Q

What are the steps in optical flow cytometry?

A

Illuminate fluid using a laser
Light scatters, emitting fluorescence
Fluorescence is collected and filtered

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11
Q

What is a laser line?

A

Single wavelength of light

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12
Q

What is the forward light scatter proportional to?

A

Size

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13
Q

What is a 90 degree light scatter proportional to?

A

Granularity

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14
Q

What can you do with a foward and 90degree light scatter?

A

Plot on a graph and identify the cell

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15
Q

How do you filter the light after scattering in optical cytometry?

A

Mirrors that separate out the light

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16
Q

What is the stokes shift?

A

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

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17
Q

What are the names of the fluorochromes?

A

Fluorescein isothiocyanate (FITC)
Phycoerythin (PE)
Peridinin chlorophyll protein (PerCP)

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18
Q

What colour does FITC emit?

A

Green

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19
Q

What colour does PE emit?

A

Orange

20
Q

What colour does PerCP emit?

A

Red

21
Q

What frequency is FITC excited and emited at?

A

Excited at 488 and emits at 520

22
Q

What frequency is PE excited and emitted at?

A

Excited at 488 and emits at 580

23
Q

What frequency is PerCP excited and emitted at?

A

Excited at 488 and emitted at 620

24
Q

What are the ideal samples for flow cytometry?

A 
Peripheral blood
Bone marrow
Fine needle aspirate
CSF and other fluids
Fresh tissue
25
Q

What are the methods of labelling in flow cytometry?

A

Direct and indirect

26
Q

How does direct labelling in flow cytometry work?

A

Monoclonal antibodies are preconjugated to fluorochromes

27
Q

How does indirect labelling in flow cytometry work?

A

Unconjugated monoclonal antibodies

28
Q

How is flow cytometry data displayed?

A

On a histogram or a box plot

29
Q

What is gating?

A

Drawing a ‘gate’ around areas of interest on a computer and then getting the computer to show just those areas in more detail

30
Q

What is cellular DNA detected using?

A

Propidium iodide

Fluorescent dye that binds preferentially to DNA

31
Q

What does propidium iodide require?

A

Permeabilisation of the plasma membrane

32
Q

At what fluorescence does propidium iodide excite and emit?

A

Excitation at 488, emits at 620

33
Q

How does propidium iodide work?

A

If it penetrates the cell membrane you can assume it to be damaged
Cells that are brightly fluorescent with it are damaged or dead

34
Q

What are some detection methods for apoptosis?

A

Propidium iodide
Phosphatidyl serine
7-aminoactinomycin D

35
Q

Where does propidium iodide peak?

A

Sub G0

36
Q

Why is propidium iodide not very reliable?

A

May detect just cell fragments and not all cell types show the sub G0 peak

37
Q

How can phosphatidyl serine be detected?

A

Incubating cells with a fluorescein labelled annexin V and propidium iodide

38
Q

Where is phosphatidyl serine normally and in apoptosis?

A

Phosphatidyl serine is normally on the inside of the cell but flips to the outside of apoptotic cells

39
Q

What does annexin V bind to?

A

Phosphatidyl serine when it is on the outside in apoptosis

40
Q

When does annexin V show up?

A

Early and late stages of apoptosis

41
Q

When does propidium iodide show up?

A

Late apoptosis and necrotic cells

42
Q

At what fluorescence is 7-aminoactinomycin excited and emitted?

A

Excited at 488 and emitted at 660

43
Q

Where does 7-aminoactinomycin intercalate?

A

G-C regions

44
Q

How do cell sorters work?

A

When the cell of interest reaches the tip of the nozzle it vibrates
-> charges the cell so it is pulled into a tube

45
Q

What are the applications of cell sorting?

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

Molecular Basis Of Disease/ Microbiology And Immunology (57 decks)

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