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Immunology: Research Practicals > Research: Flow cytometry > Flashcards

Research: Flow cytometry Flashcards

Project 3

1
Q

In the FACS Diva image of FSC-A against SSC-A (forward against side scatter). Where are the components placed?

A
  • Left corner (Low FSC and SSC): platelets and debris
  • Plateau at bottom (low SSC) but intermediate FSC (so, low internal complexity/ granularity and intermediate size): Lymphocytes: NK cells, T-cells, B-cells
  • Plateau above lymphocytes (intermediate low SSC and intermediate FSC): monocytes
  • Cloud at intermediate FSC and intermediate to high SSC above monocytes: granulocytes
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2
Q

Difference gating in histogram and scatter plot

A

In histogram: 1 variable against count
In scatter plot: 2 variables and density determines count
> example CD4 against CD8 can be done in scatter plot: 4 groups
> same sample in histogram for CD8: CD4-/+ CD- clustered together in one peak

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3
Q

Flow cytometer components
> Optics
> Fluidics
> Electronics

A

Optics
> Lasers: light source of single wavelength to generate scatter and fluorescence (excitation)
> Filters and mirrors: gather and direct the light, using mirrors and filters
> Detectors: receive the scattered or emitted light and convert it to an electronic signal
Fluidics
> Fluidics system: presents samples to the flowcell, where cells pass through the lasers one by one in a single file, using hydrodynamic focusing
Electronics
> electronics system: amplifies and converts the analog signals from detectors into digital data, every cell receives a unique value for every detector.

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4
Q

Requirements of sample for flow cytometry

A
  • Intact cells
  • Stained cells
  • Suspension of single cells
    > can be stained with immunofluorescence, but is not requirement
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5
Q

Which detector measures size? Where is it positioned?

A

Forward scatter
In line with the laser

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6
Q

Which detector measures granularity? Where is it positioned?

A

Side scatter
90 degrees relative to laser

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7
Q

Which detector measures fluorescence emission? Where is it positioned?

A

Fluorescence detector
> various positions for different fluorochromes and markers with antibody-fluorochrome conjugates
> detect emission at specific wavelengths

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8
Q

If you investigate CD4 and CD8 on cells, and choose FITC as label for the CD4 antibody. Which requirements should the second fluorochrome, for CD8 have?

A
  1. Can be excited by the lasers in the flow cytometer
  2. Emit a color that can be filtered and measured by the flow cytometer
  3. Give an emission signal that can be easily discerned from FITCs signal
    > so you want
    » minimal overlap in the emission wavelength in the spectrum
    » overlap in the excitation wavelength so that it can be excited with the same laser.
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9
Q

Fluidics system of flow cytometer

A

Cells are guided to fluidics stream > pass laser one by one > individual analysis

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10
Q

Optics: how many lasers?

A

Multiple laser send out

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11
Q

Detectors with mirrors and filters

A

Many detectors for different wavelengths after passage through mirrors and filters

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12
Q

Filter types

A
  • Longpass filters: reflects all wavelengths less than specified: LP525: > 525 nm is passed
  • Shortpass filters: reflects all wavelengths greater than specified: SP525: < 525 nm passed
  • Bandpass filters: transmits a specific band of wavelengths: BP525/50: 500-550 nm passed (25 each side)
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13
Q

Parameters of cells that can be measured for subsequent counting and maybe sorting (if FACS)

A
  • Size: FSC
  • Internal complexity / granularity: SSC
  • Markers with fluorochrome conjugated antibodies: fluorescence intensity
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14
Q

FSC

A

Forward scatter in line with laser intercept
> bigger particles generate bigger pulse

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15
Q

SSC

A

Side scatter, 90 degrees at laser intercept
> organelles, nuclei, membrane proteins, granular material affect side scatter thus granularity > stronger pulse

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16
Q

Does a 2 fold increase of FSC intensity mean a 2 fold increase in cell size?

A

No, thus intensity of pulse (FSC/SSC) is not linearly correlated to size or granularity

17
Q

Largest group in blood fraction flow cytometry

A

Granulocytes: large granularity (SSC) and intermediate size (FSC)

18
Q

If you take a certain fluorochrome, what is important for the laser and filter?

A

Laser must be in excitation spectrum
BP filter around emission spectrum to filter it out for a detector.

19
Q

Markers for B-cells, T-cells, Monocytes, NK-cells

A

B-cells: CD19
T-cells: CD3
NK-cells: CD16
Monocyte: CD14

20
Q

Fluorescnece-activated cell sorting (FACS)

A
  • Cells are broken up into droplets
  • when droplet containing cell of interest passes, a negative or positive charge is given to the droplet
  • while uncharged droplets pass, the positive and negative charges are separated and received in separate containers with electrical fields > use in further experiments
21
Q

In a scatter plot: each dot is a ..

A

Measurement (not per se a single cell)
> gating for single cells has to be done first

22
Q

Scatter plot- 4 quadrants

A
  • Double positives for two markers
  • Double negatives
  • Marker 1 positive
  • Marker 2 positive
23
Q

A histogram shows a marker negative population on the …

A

Left of the x-axis

24
Q

Advantages scatter plot and histogram

A
  • Scatter plot gives more information about characteristics of cell population: 2 parameters included
  • Histogram: better overview of number of cells at certain intensity with peaks where the highest intensity is found.
25
Q

Gating strategy: what does it mean

A

A systematic approach to select and analyze specific subsets of cells based on their fluorescence and light-scattering properties

26
Q

Gating purposes

A
  • exclude unwanted events
  • isolate for events of interest
27
Q

Gating strategy: broad and narrow gate

A

Broad gate: gating leukocytes from whole blood
Narrow gate: gate T-cells from leukocytes
Broad > Narrow gating

28
Q

Cells within the gate are … for analysis

A

Included

29
Q

Three basic steps of gating strategy

A
  1. Gate for single cells
  2. FSC vs SSC
  3. Fluorescence markers
30
Q
  1. Gate for single cells
A
  • Theoretically the flow in the flow cytometer allows cells to pass one by one, but sometimes they clumb together as doublets. > skewed results
  • look at dot plot for FSC-A against FSC-H or FSC-W
    > FSC Time against Voltage (on y-axis) plot
    » W: width
    » A: area under curve
    » H: height
    > FSC-A (x) FSC-H (y) gives diagonal line and some divergence. Include only the diagonal line: single cells: A proportional to H/W
    » if multiple cells measured at once: area of the pulse tends to be higher than height of the pulse
31
Q
  1. FSC vs SSC
A

Size vs complexity/granularity
> lymphocytes for example: intermediate to low size and low granularity > gate

32
Q
  1. Fluorescent markers
A

Add fluorescent markers distinct for cell types
> for example CD3+ cell gating to gate T-cell populations.
> Then gate within the T-cells the CD8+ CD4- T-cells: Cytotoxic T -cells (CTLs).
> also analysis of specialized markers like IFN-y within CTL gate > IFN-y expression.

33
Q

Optional: live/dead marker

A

Can be included: plotted against FSC-A on the X-axis
> live cells have low signal for the live/dead marker

Immunology: Research Practicals (4 decks)

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