Flow Cytometry Flashcards
Briefly define flow cytometry
- measures properties of a cell in flow
- sorts cells based on properties measured - Fluoresence-activated cell sorting (FACS)
What 3 things can flow cytometry can tell us about a cell?
- its relative size
- its relative granularity/internal complexity
- its relative fluorescence intensity
What are the main differences between flow cytometry and fluorescence microscopy?
- Fluorescence microscopy you’d label the surface antigen and look down a microscope, then have to scan through hundreds of fields to count the number of antigens on each cell
- with FC, you can get results from thousands of cells simultaneously - very quantitative
What are the 3 main stages of flow cytometry?
- Fluidics - cells in suspension flow in single file through an illuminated volume
- Optics - where they scatter light and emit fluorescence, and its collected and filtered
- Electronics - It is converted to digital values and stored on a computer
What are the major components of a flow cytometer?
- light source
- flow chamber
- optical system
- light detector
- computer
How do you get cells to flow down the nozzle in single file?
- need to have cells in suspension
- inject the sample into sheath fluid and pass it through a narrow tip
- sample fluid flows in a central core that doesn’t mix with sheath fluid
- introduction of a large volume into a small volume = hydrodynamic focusing
What are the two sorts of light scatter we can measure?
- Forward scatter - light that hits the sensor in a forward direction is proportional to the size
- Side scatter - light that hits and goes of a right angle is proportional to granularity
How can we measure the different wavelengths of light emitted separately?
- Light emitted passes through different filters to photomultiplying tubes
- The PMTs detect a very narrow wavelength
What is Stoke’s shift?
- The energy difference between the lowest energy peak of absorbance and the highest energy of emission
What is a fluorochrome?
- A fluorescent tag
- When it is excited by the laser, it emits light at a longer wavelength
Give the name of 3 commonly used fluorochromes and the colour light they emit
- FITC = green
- PE = Orange
- PerCP = Red
What are common sources of cells for flow cytometry?
- peripheral blood
- bone marrow
- fine needle aspirate
- fresh tissue (need to prepare in a suspension)
- CSF and other fluids
What is the direct method of labelling?
- use preconjucated monoclonal antibodies
- (flurochrome is already attached)
- e.g. if we want to quantitate the number of CD3 antigens, we use an anti-CD3 pre-conjugated with the fluorochrome
What is the indirect method of labelling?
- 2 step procedure
- e.g. use an anti-CD3 Ab, then a non-specific one that is conjugated to a fluorochrome
What are the 2 common ways of displaying the data?
Histogram or dot plot
What can we see from a histogram?
- a cell count on the y axis, and fluorescence intensity on the x axis
- gives the number of cells at a give intensity
What can we see from a dot plot?
- you can display any 2 parameters you want
- e.g. side scatter (granularity) and size (forward scatter)
What is gating?
- you can select a region on a graph that you want to investigate further - e.g. just lymphocytes
- the computer will colour these however you want, then make a dot plot of just lymphocytes in this region.
What is the advantage of using more than one fluorochrome at once?
- can quantitate lots of populations at once
- can measure many different parameters at once
- e.g. with 3, you can look at 8 populations
What molecule is commonly used to analyse cell cycle distributions?
Propidium Iodide - undergoes a dramatic increase in fluorescence upon binding DNA
How can we measure cell viability with PI?
- PI cannot normally cross the cell membrane
- if the PI penetrates the membrane, it is assumed to be damaged
- brightly fluorescing cells are damaged or dead
- The viable cell will appear negative as the PI cannot get in
Apoptosis vs Necrosis
- Apoptosis = programmed cell death, apoptotic bodies form with functioning organelles
- Necrosis = cell ruptures and releases its contents, non-functioning organelles
What does the sub-G0 peak mean?
- if cells are apoptosis, when viewing cell cycle on a histogram, there will be a peak before G0
How can we detect apoptosis?
- Phosphatidyl serine can be detected by incubating cells with Annexin-V (bound to FITC) and PI.
- PS is usually expressed on the inside of plasma membranes
- Therefore live cells wont bind the annexin as PS is inside and the PI won’t be able to get inside
- In early apoptosis, PS flips to the outside, so annexin can bind, however the PI can still not get in
- In late apoptosis, the membrane ruptures and so both can bind
- 7AAD can also bind to DNA in late apoptosis = red
- Allows all 3 stages to be quantitated
Give 5 applications of FC
- Immunophenotyping of leukaemias and lymphomas - diagnose stages with haematological neoplasm
- detection of minimal residual disease - neoplastic cell concentration is lower than 5%
- stem cell enumeration - for bone marrow transplants, count levels of CD34 in peripheral blood to see if enough bone marrow has been released following GH injections
- Organ transplant - check that there aren’t antibodies in donor or patient serum which could cause rejection
- Cd4/8 in HIV - clinically stage disease progression, measure no. of CD4+ cells because they get destroyed in HIV patient, the more CD4+ cells, the less severe case
- measurement of IC cytokines
- study of cell cycle, viability and apoptosis
- measurement of cell proliferation
- assessment of transfection efficiency
What is the difference between a cell sorting machine and flow cytometer?
- nozzle is vibrated - flow in a stream but when a charge is added, cells break off.
- these now charged cells are then sorted into positive and negative
- We can administer a charge when the cell we want is right at the end of the nozzle
- can sort two parameters at once using the different charges
What is 7AAD?
- excited by 488nm laser - emits red light at 660nm
- DNA specific - binds to G-C regions
- Used to detect apoptosis and capase activity
