Research: Flow cytometry Flashcards
Project 3
In the FACS Diva image of FSC-A against SSC-A (forward against side scatter). Where are the components placed?
- 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
Difference gating in histogram and scatter plot
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
Flow cytometer components
> Optics
> Fluidics
> Electronics
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.
Requirements of sample for flow cytometry
- Intact cells
- Stained cells
- Suspension of single cells
> can be stained with immunofluorescence, but is not requirement
Which detector measures size? Where is it positioned?
Forward scatter
In line with the laser
Which detector measures granularity? Where is it positioned?
Side scatter
90 degrees relative to laser
Which detector measures fluorescence emission? Where is it positioned?
Fluorescence detector
> various positions for different fluorochromes and markers with antibody-fluorochrome conjugates
> detect emission at specific wavelengths
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?
- Can be excited by the lasers in the flow cytometer
- Emit a color that can be filtered and measured by the flow cytometer
- 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.
Fluidics system of flow cytometer
Cells are guided to fluidics stream > pass laser one by one > individual analysis
Optics: how many lasers?
Multiple laser send out
Detectors with mirrors and filters
Many detectors for different wavelengths after passage through mirrors and filters
Filter types
- 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)
Parameters of cells that can be measured for subsequent counting and maybe sorting (if FACS)
- Size: FSC
- Internal complexity / granularity: SSC
- Markers with fluorochrome conjugated antibodies: fluorescence intensity
FSC
Forward scatter in line with laser intercept
> bigger particles generate bigger pulse
SSC
Side scatter, 90 degrees at laser intercept
> organelles, nuclei, membrane proteins, granular material affect side scatter thus granularity > stronger pulse
Does a 2 fold increase of FSC intensity mean a 2 fold increase in cell size?
No, thus intensity of pulse (FSC/SSC) is not linearly correlated to size or granularity
Largest group in blood fraction flow cytometry
Granulocytes: large granularity (SSC) and intermediate size (FSC)
If you take a certain fluorochrome, what is important for the laser and filter?
Laser must be in excitation spectrum
BP filter around emission spectrum to filter it out for a detector.
Markers for B-cells, T-cells, Monocytes, NK-cells
B-cells: CD19
T-cells: CD3
NK-cells: CD16
Monocyte: CD14
Fluorescnece-activated cell sorting (FACS)
- 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
In a scatter plot: each dot is a ..
Measurement (not per se a single cell)
> gating for single cells has to be done first
Scatter plot- 4 quadrants
- Double positives for two markers
- Double negatives
- Marker 1 positive
- Marker 2 positive
A histogram shows a marker negative population on the …
Left of the x-axis
Advantages scatter plot and histogram
- 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.
Gating strategy: what does it mean
A systematic approach to select and analyze specific subsets of cells based on their fluorescence and light-scattering properties
Gating purposes
- exclude unwanted events
- isolate for events of interest
Gating strategy: broad and narrow gate
Broad gate: gating leukocytes from whole blood
Narrow gate: gate T-cells from leukocytes
Broad > Narrow gating
Cells within the gate are … for analysis
Included
Three basic steps of gating strategy
- Gate for single cells
- FSC vs SSC
- Fluorescence markers
- Gate for single cells
- 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
- FSC vs SSC
Size vs complexity/granularity
> lymphocytes for example: intermediate to low size and low granularity > gate
- Fluorescent markers
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.
Optional: live/dead marker
Can be included: plotted against FSC-A on the X-axis
> live cells have low signal for the live/dead marker
