Flow Cytometry

Question 1

Briefly define flow cytometry

Answer 1

• measures properties of a cell in flow

- sorts cells based on properties measured - Fluoresence-activated cell sorting (FACS)

Question 2

What 3 things can flow cytometry can tell us about a cell?

Answer 2

• its relative size
• its relative granularity/internal complexity
• its relative fluorescence intensity

Question 3

What are the main differences between flow cytometry and fluorescence microscopy?

Answer 3

• 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

Question 4

What are the 3 main stages of flow cytometry?

Answer 4

• 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

Question 5

What are the major components of a flow cytometer?

Answer 5

• light source
• flow chamber
• optical system
• light detector
• computer

Question 6

How do you get cells to flow down the nozzle in single file?

Answer 6

• 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

Question 7

What are the two sorts of light scatter we can measure?

Answer 7

• 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

Question 8

How can we measure the different wavelengths of light emitted separately?

Answer 8

• Light emitted passes through different filters to photomultiplying tubes
• The PMTs detect a very narrow wavelength

Question 9

What is Stoke’s shift?

Answer 9

• The energy difference between the lowest energy peak of absorbance and the highest energy of emission

Question 10

What is a fluorochrome?

Answer 10

• A fluorescent tag

- When it is excited by the laser, it emits light at a longer wavelength

Question 11

Give the name of 3 commonly used fluorochromes and the colour light they emit

Answer 11

• FITC = green
• PE = Orange
• PerCP = Red

Question 12

What are common sources of cells for flow cytometry?

Answer 12

• peripheral blood
• bone marrow
• fine needle aspirate
• fresh tissue (need to prepare in a suspension)
• CSF and other fluids

Question 13

What is the direct method of labelling?

Answer 13

• 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

Question 14

What is the indirect method of labelling?

Answer 14

• 2 step procedure

- e.g. use an anti-CD3 Ab, then a non-specific one that is conjugated to a fluorochrome

Question 15

What are the 2 common ways of displaying the data?

Answer 15

Histogram or dot plot

Question 16

What can we see from a histogram?

Answer 16

• a cell count on the y axis, and fluorescence intensity on the x axis
• gives the number of cells at a give intensity

Question 17

What can we see from a dot plot?

Answer 17

• you can display any 2 parameters you want

- e.g. side scatter (granularity) and size (forward scatter)

Question 18

What is gating?

Answer 18

• 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.

Question 19

What is the advantage of using more than one fluorochrome at once?

Answer 19

• can quantitate lots of populations at once
• can measure many different parameters at once
• e.g. with 3, you can look at 8 populations

Question 20

What molecule is commonly used to analyse cell cycle distributions?

Answer 20

Propidium Iodide - undergoes a dramatic increase in fluorescence upon binding DNA

Question 21

How can we measure cell viability with PI?

Answer 21

• 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

Question 22

Apoptosis vs Necrosis

Answer 22

• Apoptosis = programmed cell death, apoptotic bodies form with functioning organelles
• Necrosis = cell ruptures and releases its contents, non-functioning organelles

Question 23

What does the sub-G0 peak mean?

Answer 23

• if cells are apoptosis, when viewing cell cycle on a histogram, there will be a peak before G0

Question 24

How can we detect apoptosis?

Answer 24

• 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

Question 25

Give 5 applications of FC

Answer 25

- 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

Question 26

What is the difference between a cell sorting machine and flow cytometer?

Answer 26

- 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

Question 27

What is 7AAD?

Answer 27

- excited by 488nm laser - emits red light at 660nm - DNA specific - binds to G-C regions - Used to detect apoptosis and capase activity