Flow Cytometry

Question 0

Explain hydrodynamic focusing

Answer 0

The process where the cells are ordered into a single stream of particles using high pressure in the sample stream (through pressurised laminar flow), so that one cell flows past the laser at a given time

Question 1

Outline the 3 main systems in a flow cytometer.

Answer 1

Fluidics: deliver particles of interest to the laser for interrogation
Optics: provides excitation sources (lasers) to illuminate the particles in the sample stream and the optical filters to direct the reflected light to the appropriate detectors
Electronics: convert light signals to equivalent electronic signals

Question 2

How do you control the diameter of the sample core?

Answer 2

By using the sample pressure regulator to adjust the pressure of the sample core: higher pressure = smaller diameter

Question 3

List the commonly used lasers and their wavelengths:

Answer 3

Argon: 488nm, blue
UV: 405nm, violet
Helium neon (He-Ne): 640nm, Red
He-Ne: 543nm, Green

Question 4

Outline the 2 types of detectors used in Flow Cyt:

Answer 4

Photodiodes: used for strong signals, measures light scatter

Photomultiplier tubes: used for weaker signals, measures fluorescence

Question 5

Outline the 3 types of optical filters:

Answer 5

Bandpass: allows a specific range of wavelength of light through
Longpass: allows wavelengths equal to or longer than the designated to pass, and filters out the shorter ones
Shortpass: allows wavelengths equal to or shorter than the designated to pass, filtering out the longer ones

Question 6

What are the properties measured using Flow Cyt?

Answer 6

Physical: relative size and internal complexity
Chemical: relative fluorescence intensity

Question 7

What are the two types of staining methods using antibodies?

Answer 7

Direct: Ab directly conjugated to a fluorophore/fluorochrome
Indirect: Primary Ab unconjugated, followed by secondary Ab binding by an Ab conjugated to a fluorochrome

Question 8

Outline the common types of fluorochromes.

Answer 8

-Classical dyes: stable, broad selection, eg FITC, PE, PerCP, APC
-Alexa Fluor dyes: stable, bright, less pH sensitive
-Pacific dyes: dim
-Tandem dyes: resonance energy transfer dyes, covalently-linked, eg PE-Cy5, unstable (sensitive to light, temp and chemicals)
-Quantum dots: semiconductor nanoparticles, inorganic crystal cores coated with zinc sulfide, core size determines wavelength, narrow spectrum
Brilliant Violet Dyes: fluorescent polymer probes, high intrinsic brightness, little non-specific binding, stable to fixation

Question 9

What is the Staining Index (SI)?

Answer 9

It is a measure of the overall brightness of the fluorophores by minusing the the negative (background) signals from the positive signals, taking into account their SD’s

Question 10

Explain the use and types of viability dyes.

Answer 10

Used to label dead cells; they are problematic as they could cause more autofluorescence by binding non-specifically to Ab
DNA-binding dyes: binds DNA where the (dead) cell membranes are compromised.
Protein-binding dyes: increased fluorescence in dead cells as more dye will reach inside and bind to proteins inside cell (instead of only binding to surface proteins)

Question 11

What is a voltage pulse?

Answer 11

It is the representation of light signal in voltage units, which can be quantified.

Question 12

Outline the “list-mode” format of data storage.

Answer 12

Each event has a value for each parameter measured, eg a value for FITC and PE, and plotted on the 2D plot as a dot

Question 13

What determines the fluorescence intensity?

Answer 13

Emitted fluorescent light intensity is proportional to the number of copies of the labelled protein

Question 14

What are the different types of data display available?

Answer 14

Single display: Histogram

Dual-parameter display: Pseudo-colour, contour, zebra, Density

Question 15

Explain the importance of Compensation.

Answer 15

The need for compensation arises when the emission spectra of the fluorophores overlap, meaning eg a PE PMT may pick up fluorescence emitted from the FITC, as there is a FITC tail overlapping the PE spectrum. We therefore need compensation to correct this, by using a control (only 1 fluorophore used, eg only FITC, we can see how much fluorescence is detected in PE), and minus the uncompensated fluorescence from the actual data, thus compensating the data. In the control, the neg-median of the unstained PMT (PE) must be equal to the pos-median. (We are expecting no signal in this PMT, tf the neg peak (median) should be the same as the positive median (positive for FITC, but not PE).

Question 16

What are some of the considerations to be aware of for Immunophenotyping?

Answer 16

• Activated cells can increase or decrease some protein levels on or inside the cells
• Freeze-thawing or cell isolation techniques can change the protein expression
• Enzyme Rx to dissociate cells or fixation of cells can lead to shearing off of epitopes
• Non-specific Ab binding