Flow Cytometry- Introduction And Applications

Question 1

What is flow cytometry?

Answer 1

A technique that simultaneously measures several physical characteristics belonging to a single cell in suspension

Question 2

What is the definition of flow cytometry?

Answer 2

Measuring the properties of cells in flow

Question 3

What is flow sorting?

Answer 3

Sorting (separating) cells based on properties measured in flow

Question 4

What is another name for flow sorting?

Answer 4

Fluorescence-activated cell sorting (FACS)

Question 5

What can a flow cytometer tell us about a cell?

Answer 5

Relative size,
relative granularity/ internal complexity,
Relative fluorescence intensity

Question 6

What can we measure with a flow cytometer?

Answer 6

Surface receptors
Intracellular cytokines/enzymes
Measure cell cycle viability and apoptosis

Question 7

What are fluidics?

Answer 7

Cells in suspension flowing in a single file

Question 8

How do fluidics work?

Answer 8

Inject a sample into a sheath fluid as it passes through a small hole
Simple fluid flows down a central core

Question 9

What is hydrodynamic focusing?

Answer 9

Introduction of a large volume into a smaller volume

Question 10

What are the steps in optical flow cytometry?

Answer 10

Illuminate fluid using a laser
Light scatters, emitting fluorescence
Fluorescence is collected and filtered

Question 11

What is a laser line?

Answer 11

Single wavelength of light

Question 12

What is the forward light scatter proportional to?

Answer 12

Size

Question 13

What is a 90 degree light scatter proportional to?

Answer 13

Granularity

Question 14

What can you do with a foward and 90degree light scatter?

Answer 14

Plot on a graph and identify the cell

Question 15

How do you filter the light after scattering in optical cytometry?

Answer 15

Mirrors that separate out the light

Question 16

What is the stokes shift?

Answer 16

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

Question 17

What are the names of the fluorochromes?

Answer 17

Fluorescein isothiocyanate (FITC)
Phycoerythin (PE)
Peridinin chlorophyll protein (PerCP)

Question 18

What colour does FITC emit?

Answer 18

Green

Question 19

What colour does PE emit?

Answer 19

Orange

Question 20

What colour does PerCP emit?

Answer 20

Red

Question 21

What frequency is FITC excited and emited at?

Answer 21

Excited at 488 and emits at 520

Question 22

What frequency is PE excited and emitted at?

Answer 22

Excited at 488 and emits at 580

Question 23

What frequency is PerCP excited and emitted at?

Answer 23

Excited at 488 and emitted at 620

Question 24

What are the ideal samples for flow cytometry?

Answer 24

Peripheral blood
Bone marrow
Fine needle aspirate
CSF and other fluids
Fresh tissue

Question 25

What are the methods of labelling in flow cytometry?

Answer 25

Direct and indirect

Question 26

How does direct labelling in flow cytometry work?

Answer 26

Monoclonal antibodies are preconjugated to fluorochromes

Question 27

How does indirect labelling in flow cytometry work?

Answer 27

Unconjugated monoclonal antibodies

Question 28

How is flow cytometry data displayed?

Answer 28

On a histogram or a box plot

Question 29

What is gating?

Answer 29

Drawing a ‘gate’ around areas of interest on a computer and then getting the computer to show just those areas in more detail

Question 30

What is cellular DNA detected using?

Answer 30

Propidium iodide | Fluorescent dye that binds preferentially to DNA

Question 31

What does propidium iodide require?

Answer 31

Permeabilisation of the plasma membrane

Question 32

At what fluorescence does propidium iodide excite and emit?

Answer 32

Excitation at 488, emits at 620

Question 33

How does propidium iodide work?

Answer 33

If it penetrates the cell membrane you can assume it to be damaged Cells that are brightly fluorescent with it are damaged or dead

Question 34

What are some detection methods for apoptosis?

Answer 34

Propidium iodide Phosphatidyl serine 7-aminoactinomycin D

Question 35

Where does propidium iodide peak?

Answer 35

Sub G0

Question 36

Why is propidium iodide not very reliable?

Answer 36

May detect just cell fragments and not all cell types show the sub G0 peak

Question 37

How can phosphatidyl serine be detected?

Answer 37

Incubating cells with a fluorescein labelled annexin V and propidium iodide

Question 38

Where is phosphatidyl serine normally and in apoptosis?

Answer 38

Phosphatidyl serine is normally on the inside of the cell but flips to the outside of apoptotic cells

Question 39

What does annexin V bind to?

Answer 39

Phosphatidyl serine when it is on the outside in apoptosis

Question 40

When does annexin V show up?

Answer 40

Early and late stages of apoptosis

Question 41

When does propidium iodide show up?

Answer 41

Late apoptosis and necrotic cells

Question 42

At what fluorescence is 7-aminoactinomycin excited and emitted?

Answer 42

Excited at 488 and emitted at 660

Question 43

Where does 7-aminoactinomycin intercalate?

Answer 43

G-C regions

Question 44

How do cell sorters work?

Answer 44

When the cell of interest reaches the tip of the nozzle it vibrates -> charges the cell so it is pulled into a tube

Question 45

What are the applications of cell sorting?

Answer 45

``` Immunophenotyping of leukaemias and lymphomas Detection of MRD Stem cell enumeration CD4/CD8 in HIV Measurement of intracellular cytokines Study of cell cycle, viability and apoptosis Measurement of cell proliferation Assessment of transfection efficiency ```