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AP0609 Component 1 > Flow cytometry and Cell sorting > Flashcards

Flow cytometry and Cell sorting Flashcards

1
Q

What are the main benefits of flow cytometry?

A
  • The simultaneous measurement of multiple physical characteristics of single cells, one at a time, in a cell suspension
  • High throughput
  • Measurements are made on a per cell basis at rates typically in the order of 500 to 20,000 cells per second in a moving fluid stream
  • Alot of data very quickly, allowing you to find very rare cells
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2
Q

What are the 3 requirements for flow cytometry?

A
  • Fluidics
  • Optics
  • Electronics
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3
Q

What is the order of events in a flow cytometer?

A

Laser - Fluid - Fluorescence - Filters - Electronics - Quantification

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4
Q

Describe the concept of fluidics in flow cytometry

A
  • Delivery of individual particles/cells to a specific point intersected by a laser beam
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5
Q

What is Sheath Fluid?

A

Sheath fluid is filtered isotonic saline, or phosphate buffered saline, to allow cells to keep there morphology.
Sheath fluid ensures laminar coaxial flow.

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6
Q

Name some examples of samples that could be used in flow cytometry

A
  • White blood cells
  • Processed tissue
  • Cells in culture
  • Bone Marrow
  • Beads
  • Bacteria
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7
Q

What is the issue of using whole blood in Flow cytometry?

A

There is too many red blood cells in a whole blood sample which will affect the result , therefore red blood cells are lysed in a salt solution.

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8
Q

Describe the Bernoulli Effect.

A

When the velocity of the fluid is slower on the sides of the tube due to drag. The fluid moves towards low pressure. This means that there is no turbulence in the flow and that the cells are single file.

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9
Q

What velocity is needed to ensure laminar flow and hydrodynamic focusing?

A

Approximately 10 meters/sec. Meaning 10 micron particles will transverse there own diameter in 1 microsecond.

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10
Q

What are the 4 components of the optics in a flow cytometer?

A
  • Excitation Source
  • Fluorescence
  • Collection optics - filters/mirrors
  • Detectors
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11
Q

Why do we use LASERs in flow cytometry?

A
  • Produce a coherent, plane polarized, intense, narrow beam of light
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12
Q

How does a LASER work?

A

Plasma tube contains a gas under pressure which fluoresces under the amplification of the current. When the photons strike an atoms in an excited state they release another photon of the same wavelength.

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13
Q

What is the physics of fluorescence?

A

Excitation of a fluorochrome - when it receives the excitation its energy state increases and then very rapidly drops the energy state and at that point emits light at a longer wavelength.

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14
Q

Why use Tandem dyes?

A

Individual fluorochromes that are bound together can broaden the scope creating different wavelengths.

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15
Q

What are the 3 components of the optics in a flow cytometer?

A
  • Beam mixer - takes lasers from multiple sources into one specific direction
  • Filters/mirrors - angle light to the detectors often for specific wavelengths
  • Detectors - Photodiodes, Photomultiplier tubes (PMT)
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16
Q

What are the 4 types of Filters/mirrors?

A

Dichroic mirrors - Allow light of a certain wavelength to be reflected while the remaining wavelength passes through
- Shortpass filters - Allow a light below a specific wavelength through
- Bandpass filters - Only allow a range of wavelengths through
- Longpass filters - Allow a light above a specific wavelength through

17
Q

What are the positives and negatives of PMTs?

A
  • Most common detector
  • Adjustable
  • High Sensitivity
  • Poor efficiency in red
  • Inexpensive
18
Q

What are the positives and negatives of photodiodes?

A
  • Newer technology
  • High efficiency
  • No adjustable sensitivity
19
Q

What are the 3 steps of the electronics system in flow cytometry?

A

Amplification - Analog to digital converter - Computer system

20
Q

What is compensation?

A

Spectral overlap can occur so compensation removes cells fluorescing in the wrong channel

21
Q

What does Forward Scatter show (FS)

A

Size of the cells

22
Q

What does Side Scatter show (SS)

A

Internal structures of the cells i.e. granularity

Dead cells can also have a high side scatter due to there rough surface

23
Q

What are the two ways to display the results from flow cytometry?

A
  • Frequency histogram
  • Dot Plot
24
Q

What do the locations on a dot plot show?

A

Upper left -+
Upper right ++
Lower left –
Lower right +-

25
Q

What is a threshold?

A

A threshold can be used to remove any residual dead or red blood cells for example and they therefore will not be analysed

26
Q

What is the use of Gating?

A

You can gate (select) specific cells from and initial analysis and these gated cells can be analysed for further parameters

27
Q

What are the benefits of multi-colour flow cytometry?

A
  • More accurate dure to the ability to gather more information
  • Can use smaller amounts of sample due to more parameters per tube
  • Saves time and reagents
28
Q

What is multiplex flow cytometry?

A

Where you measure any analytes simultaneously
It has the same benefits as multi colour flow cytometry
Luminex is an example

29
Q

Describe Luminex technology

A
  • Uses microspheres to which reagents can bind to
  • Each microsphere is dyed to give a unique signature - antibodies are bound to the beads
  • Beads are then incubated with the sample where the analyte is captured on the beads
30
Q

What are the 2 ways cell sorting can work?

A
  • Electrostatic deflection of a stream of air
  • Mechanical cells sorting within the flow cell
31
Q

Describe the process of electrostatic deflection

A
  • Each droplet through the flow cell is given a charge.
  • These droplets then flow passed positive and negative elctrode.
  • If the droplets are poitive the with move towards the negative electrode and go to one tube
  • If the droplet is negative then is will move towards the positive electrode and go into that tube
32
Q

What factors can effect electrostatic deflection cell sorting?

A
  • Temperature
  • Pressure
33
Q

Describe the process of mechanical cell sorting

A
  • Mechanical cell sorting is when in the flow cell there is a physical catcher tube and this tube mechanically moves in and out of the flow of the cells to capture the wanted cells

AP0609 Component 1 (7 decks)

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