Introduction To Flow Cytometry Flashcards
- What is flow cytometry?
o Technique which simultaneously measures several physical characteristics belonging to a single cell in suspension.
o This is done by light scatter and fluorescence.
o Flow Cytometry: Measuring properties of cells in flow
- What is Flow Sorting aka Fluorescence-Activated Cell Sorting (FACS):
Sorting (separating) cells based on properties measured in flow.
- What can flow cytometry tell us about a cell?
- > Relative Size
- > Relative Granularity/internal Complexity
- > Relative Fluorescence Intensity
- > Cytokines,Adhesion,Surface Receptors
- What are two methods of visualisation of cells?
- Fluorescence Microscopy
2. Flow Cytometry
- What are some disadvantages of fluorescence Microscopy?
- > Only looks at cells within specific field- need many fields to spot rare cells
- > Not quantitive
- > When results show variable fluorescence , its subjective
- What is the basic principle of flow cytometry?
o Fluidics: cells in a suspension that flow in a single-file.
o Optics: laser hits the illuminated volume of cells where light is scattered, and fluorescence emitted.
o Electronics: fluorescence is scattered, filtered and converted to digital values that are stored on a computer.
o Light source -> flow chamber -> optical system -> light detectors -> computer.
- In the process of Fluidics , the cells in the suspension have to flow in a single file, how do we achieve this?
-> Injecting the sample into the sheath fluid as it passes through a small (50-300um) orrifice .
(By making it go through such a narrow passage, only one cell can fit at a time and so they’re single file)
- How do we make sure that their is a laminar flow in the cytometry?
Sample fluid flows in a central core that does not mix with the sheath fluid: this creates laminar flow (smooth, non-turbulent flow).
- What is Hydrodynamic Focusing?
by building up the walls of the tunnel from fluid, using the effects of fluid dynamics. A wide tube is used, through which a “wall” of fluid called the sheath flow is pumped. The sample is injected into the middle of the sheath flow. If the two fluids differ enough in their velocity or density, they do not mix: they form a two-layer stable flow.
Large Vol–>Small Vol
- In the process of Optics .:
What type of wavelength of laser light do we use?
We usually use a single wavelength of light (a laser line) or less commonly we use a mixture of wavelengths
- Fill in the blank:
can provide light from ** to *, and can provide coherent, ** frequency light
can provide light from milliwatts to watts, and can provide coherent, single frequency light
- Are the lasers used in flow cytometry expensive?
can be inexpensive, air-cooled units or expensive, water-cooled units
- What is the difference between forward and side scatter?
o When light interacts with non-fluorescent (not labelled cells), the light is scattered in 2 directions.
- Forward scatter: proportional to the size of the RBC.
- Side scatter: proportional to the granularity or internal complexity of cells.
- Rank the following WBC’s:
-Lymphocytes
-Neutrophils
-Monocytes
in terms of size and complexity
Size:
- Monocytes-Biggest
- Neutrophils
- Lymphocytes-Smalles
Complexity:
- Neutrophils-Most
- Monocytes
- Lymphocytes-Least
15.Explain the channel layout for laser based flow cytometry
o When light hits the fluorescence labelled cells (labelled with 4 antibodies that each have a different colour), light is emitted.
o This light passes through filters and mirrors (due to the overlap in the different colours) before light of a specific wavelength is picked up by photomultiplier tubes.
- Label a simple diagram of a flow cytometry?
I cant put pictures on here but go label one innit
- Label a simple diagram of the channel layout in a flow cytometer?
I cant put pictures on here but go label one innit
- What is Analog-Digital Conversation?
Does what it says , processes signals from detectors into a digital/electronic form
- What is a fluorescence stokes shift?
o When fluorophores are hit with fluorochromes e.g. FITC, they absorb fluorescence to become excited but then emit fluorescence to return to their unexcited state again.
o The energy difference between the lowest energy peak of absorbance and the highest energy of emission.
20. In Immunofluorescence we use fluorochromes and dyes Which fluorochromes with the colours: -Green -Orange -Red?
o Fluorescein isothiocyanate (FITC) – Green (520nm).
o Phycoerythrin (PE) – Orange (580nm)
o Peridinin Chlorophyll Protein (PerCP) – Red (620).
- Where would we get the single cells in the suspension?
Peripheral blood Bone marrow Fine Needle Aspirate CSF and other fluids Fresh Tissue
- What does MoABS stand for?
Monoclonal Antibodies
- What is the difference between direct and indirect immunofluorescence labelling?- SCIENCY way
o Direct: monoclonal antibodies (MoABs) are pre-conjugated (directly labelled) to fluorochromes.
o Indirect: unconjugated MoABs that other labelled antibodies bind to.
- What is the difference between direct and indirect immunofluorescence labelling?- SIMPLE way
- Basically Direct just has a cell, its antigen is bound to a primary antibody that will directly bind to fluorophore
- Indirect has a primary antibody which binds to a few secondary antibodies which already have the fluorophore on it
- Whats a disadvantage of the indirect immunofluorescence method?
A lot of background staining
- Following questions are about histograms:
-How many parameters?
-What does the X axis represent
-What does the Y axis represent?
-
- One parameter
- X axis = Fluorescence Intensity
-Y axis = Cell count
Can only measure one parameter at a time - eg how many cells are fluorescent/Bright
27. Following questions are about Dot Plots: -How many parameters? -What does the X axis represent -What does the Y axis represent? -
-Two parameters
-X axis = Side scatter
-Y axis = Forward Scatter
Quantitate cells on basis of two parameters- the axis can be anything eg fluorescence
- What is gating?
o Regions of cells with characteristics are ‘gated’/selected (draw a gate around them) so that we can look at specific populations of cells.
- What Analysis would you do after gating on the computer?
o To quantitate the gated information.
o Machine quantitates the information.
- If blood is labelled with 2 markers how can 4 populations of cells be identified using the 2 step dot plot?
Okay so say you label blood with PE and FITC:
- A population of positive for PE
- A population for positive for FITC
- A Double positive situation -for both PE and FITC
- A negative population for neither PE or FITC
- How many populations can we identify with 3 antibodies?
8
eg FITC, PE and PerCP , 8 diff combinations of when they’re positive or negative
