Research: Flow cytometry

Question 1

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

Answer 1

• 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

Question 2

Difference gating in histogram and scatter plot

Answer 2

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

Question 3

Flow cytometer components
> Optics
> Fluidics
> Electronics

Answer 3

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.

Question 4

Requirements of sample for flow cytometry

Answer 4

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

Question 5

Which detector measures size? Where is it positioned?

Answer 5

Forward scatter
In line with the laser

Question 6

Which detector measures granularity? Where is it positioned?

Answer 6

Side scatter
90 degrees relative to laser

Question 7

Which detector measures fluorescence emission? Where is it positioned?

Answer 7

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

Question 8

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?

Answer 8

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.

Question 9

Fluidics system of flow cytometer

Answer 9

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

Question 10

Optics: how many lasers?

Answer 10

Multiple laser send out

Question 11

Detectors with mirrors and filters

Answer 11

Many detectors for different wavelengths after passage through mirrors and filters

Question 12

Filter types

Answer 12

• 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)

Question 13

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

Answer 13

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

Question 14

FSC

Answer 14

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

Question 15

SSC

Answer 15

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

Question 16

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

Answer 16

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

Question 17

Largest group in blood fraction flow cytometry

Answer 17

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

Question 18

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

Answer 18

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

Question 19

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

Answer 19

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

Question 20

Fluorescnece-activated cell sorting (FACS)

Answer 20

• 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

Question 21

In a scatter plot: each dot is a ..

Answer 21

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

Question 22

Scatter plot- 4 quadrants

Answer 22

• Double positives for two markers
• Double negatives
• Marker 1 positive
• Marker 2 positive

Question 23

A histogram shows a marker negative population on the …

Answer 23

Left of the x-axis

Question 24

Advantages scatter plot and histogram

Answer 24

• 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.

Question 25

Gating strategy: what does it mean

Answer 25

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

Question 26

Gating purposes

Answer 26

- exclude unwanted events - isolate for events of interest

Question 27

Gating strategy: broad and narrow gate

Answer 27

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

Question 28

Cells within the gate are ... for analysis

Answer 28

Included

Question 29

Three basic steps of gating strategy

Answer 29

1. Gate for single cells 2. FSC vs SSC 3. Fluorescence markers

Question 30

1. Gate for single cells

Answer 30

- 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

Question 31

2. FSC vs SSC

Answer 31

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

Question 32

3. Fluorescent markers

Answer 32

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.

Question 33

Optional: live/dead marker

Answer 33

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