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Honours Exam LTC > Flow Cytometry > Flashcards

Flow Cytometry Flashcards

0
Q

Objective of fluidics subsystem

A

One cell moving past laser beam at a particular time.
Pressurized laminar flow (hydrodynamic focusing)
Injected through sheath fluid

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1
Q

Three subsystems of flow cytometry

A

Fluidics
Optics (excitation and collection)
Electronics

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2
Q

What does laser stand for

A

Light amplification by stimulated emission of radiation

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3
Q

Two main types of photodetectors

A

Photodiodes - strong signals

Photomultiplier tubes - more sensitive, small fluorescence

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4
Q

What is the role of optical filters

A

Determine specificity of detection

Long-,short- and band-pass

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5
Q

Flow cytometric measurements

A

Physical - relative size/ internal complexity

Chemical - relative fluorescent intensity

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6
Q

Forward scatter related to

A

Cell size

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7
Q

Side scatter related to

A

Cellular granularity and internal complexity

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8
Q

What is a fluorochrome

A

Organic molecule which accepts light energy (excitation) at a given wavelength and re-emits (emission) at a longer wavelength.

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9
Q

Critical aspects of staining for FC

A

Ab non-specificity (
Appropriate controls (ns binding - stain with 2ndry Ab only, background - don’t stain)
Ab titrations

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10
Q

Explain stokes shift

A

Ground state molecule is excited. Energy is given off in form of heat. Remaining energy is emitted at longer wavelength light.

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11
Q

Types of fluorochromes

A 
Classic dyes
Alexa Fluor / brilliant violet
Pacific dyes
Tandem dyes
Quantum dots
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12
Q

How is light converted to data

A

Photodetection
Amplification
Signal processing (quantify)

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13
Q

What is used for dual and single parameter display

A

Dotplot and histogram

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14
Q

Why do we need compensation in FC

A

Emission at range of wavelengths. Emission spectra tail extends to longer wavelengths leading to SPECTRAL OVERLAP = detection in more than one detector

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15
Q

What is compensation

A

Removal of background signal in other channels. Calculates the amount of interference a fluorochrome will have in a channel.

16
Q

What is the goal of compensation

A

For a given channel, the median signal of both positive and negative populations in other channels should be the same.

17
Q

Practical considerations when running FC

A

Ensure PMT voltages are correct
Compensation before acquiring
Make singly stained controls

18
Q

How does one select population of interest in FC

A

Gating

19
Q

Name types of gating

A

Polygon
Quadrant
Histogram (interval)

20
Q

What is a CFSE assay

A

Carboxyfluorescein succinimidyl ester
Diffuses into cells, cleaved by esterases to for membrane impermeable fluorescent dye. Cells given stimulus. As cells divide signal in each cell halves.

21
Q

Components of cell sorter

A 
Interrogation point
Charging stream
Droplet formation
Electrical plates
Waste collection
Collection vessels

Honours Exam LTC (10 decks)

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