9- Flow Cytometry

Question 1

what is flow cytometry?

Answer 1

measuring (physical and chemical) properties of cells in flow

Question 2

what is flow sorting or FACs?

Answer 2

FACS = fluorescence activated cell sorting

the (physical) separation of cells based on properties measured in flow

Question 3

what can flow cytometry tell us about a cell?

Answer 3

relative size

relative granularity

relative fluorescence activity (by adding a fluorescent marker) = can observe activity of DNA, cell surface receptors, intracellular components…

Question 4

advantages of flow cytometry

Answer 4

analysis 1000s of cells per second = automated system, high-throughput analysis for large cell populations

quantitative date on cell characteristics

can detect subtle differences in fluorescence intensity amongst cells & identify rare cells

Question 5

what are the main components of flow cytometry?

Answer 5

light source
cells in suspension

fluidics - flow chamber

optics - optical system with laser as light source, and lenses, filters and mirrors

electronics - light detectors/PMTs and computer system

Question 6

what do PMTs do?

Answer 6

photomultiplier tubes - act as light detectors

they’re sensitive to low light levels, and are positioned to detect scattered light in forward and sideways directions = converted into electrical signal

Question 7

describe the fluidics of the flow chamber

Answer 7

cells in suspension are introduced into the flow chamber - enter a laminar, single-file, central flow through the chamber to ensure each cell passes through the laser individually

single-file central flow is ensured by sheath fluid running alongside flow chamber - ensures laminar flow and hydrodynamic focusing

Question 8

describe the optics of the laser and additional equipment

Answer 8

involves laser, lenses, filters and mirrors

laser directs a specific light wavelength at each cell in single-file laminar flow individually

light is directed onto cells by lenses, mirrors and filters

cells scatter the light - forwards scatter is proportional to cell size, sideways scatter for relative granularity

Question 9

describe the electronics of flow cytometry

Answer 9

involves PMT tubes and computer software/ analysis

PMT tubes collect scattered light in forwards and sideways directions - converts detected light signals into electrical signals

signals are then processed by computer software into measurable data on relative cell size and granularity

Question 10

describe flow cytometry with fluorophores

Answer 10

cells are labelled with fluorophore-conjugated antibodies = different antibodies bind to their specific cell surface markers, and different antibodies are labelled with different fluorophores that emit light at a specific wavelength

individual cells pass through flow cell in single file - individually targeted by a laser which emits light at a specific wavelength

light hits the cells and scatters in forwards & sideways directions

dichroic filters are placed strategically across the detection path of scattered light to PMTs- different PMTs have different dichroic filters in front of them that match the emission wavelength of the fluorophores

PMTs receive light within a narrow wavelength range - received light signals are converted into electrical signals

computer software converts electric signals into measurable data for analysis, visualisation and interpretation

Question 11

purpose of the dichroic filter?

Answer 11

very accurate colour filter used to selectively pass light of a small wavelength range/ colour range

Question 12

what is the advantage of fluorophore-added flow cytometry?

Answer 12

different fluorochromes emit light at distinct wavelengths and have corresponding filters

allows for multi-parametric analysis of individual cells = measurements of cell size, granularity and specific cell surface markers

allows for discrimination between various cell popls within a heterogenous sample

Question 13

what is a fluorophore?

Answer 13

a molecule that can absorb light at one wavelength and emit wavelength at a different, longer wavelength

Question 14

what is fluorescence?

Answer 14

when a substance absorbs light at one wavelength (excitation wavelength) and then emits light at a longer wavelength (emission wavelength) when returning to an unexcited state

Question 15

what is the Stokes shift?

Answer 15

the difference in wavelength between the excited/absorbed and emission/ emitted light in fluorescence

Question 16

describe fluorescence with emission and excitation spectrum wavelengths

Answer 16

excitation spectrum is the range of light wavelengths a fluorophore can absorb
- peak is the wavelength at which light is absorbed most efficiently

emission wavelength is the range of light wavelengths a fluorophore emits after being excited - returns to unexcited state
- peak is the wavelength at which emitted light is the most intense

emission light wavelength is typically at a longer wavelength than excitation wavelength - excitation first, then emission

difference between excitation and emission = Stokes Shift

Question 17

list some commonly used fluorochromes and the colours they emit

Answer 17

FITC - green
PE - orange
PerCP - red

Question 18

explain why combinations of fluorochromes can be used together, and the benefits of such use of combinations of fluorochromes.

Answer 18

different fluorochromes have distinct emission wavelengths

different antibodies labelled with different fluorochromes can target specific cell surface markers

• allows for multiplexing/ multi-parametric analysis, and discrimination between various cell populations in a heterogenous ample

Question 19

what is immunofluorescence?

Answer 19

technique that uses the binding specificity of antibodies and antigen - allows for detecting various target biomolecules within a cell quantitatively

Question 20

what are the two methods for immunofluorescence?

Answer 20

direct - monoclonal antibodies are conjugated directly to fluorochromes

indirect - fluorochromes are attached to secondary antibodies
= primary antibody binds to target antigen, secondary antibody with fluorochrome binds to first antibody

Question 21

compare direct and indirect immunofluorescence

Answer 21

direct - cleaner, quicker way of staining

indirect - less clean, more background noise, more intermediary steps

Question 22

what dye is used to analyse cell DNA?

Answer 22

propidium iodide - emits a red fluorescence; excited at 400nm, emits at wavelength 600nm

Question 23

list three different parameters that can be measured from cell DNA using flow cytometry

Answer 23

• cell cycle = proportion of cells in each stage
• cell viability = healthy living cells vs dead/dying cells
• measuring apoptosis

Question 24

describe analysis of cell DNA using PI and flow cytometry

Answer 24

propidium iodide (PI) is commonly used - it’s a fluorescent dye that binds preferentially to DNA

cell needs to be permeabilised to let dye in and then fixed to maintain cell structure and DNA within

PI dye is excited at 300nm, emits red fluorescence at 600nm
- fluorescence emitted is proportional to the amount of DNA used

Question 25

describe what is expected in DNA analysis of the cell cycle within a cell population

Answer 25

larger proportion of cells within G0/1 with 2N haploid content

decrease in cell proportion in S phase - between 2N and 4N as cell contents/DNA is being replicated

smallest proportion of cells in G2/M phase - cells have 4N content, have completed DNA replication but haven’t undergone mitosis

Question 26

describe the analysis of cell DNA viability in flow cytometry (healthy vs living cells)

Answer 26

healthy living cells will have intact cell membranes
= PI/ propidium iodide can’t cross, cells will exhibit little to no fluorescence

dead/damaged cells will have compromised membrane integrity
= PI can cross and bind to DNA

fluorescence increases the more PI interacts with cellular DNA - cells passed through flow cell, fluorescence detected by PMPTs and a scatterplot or histogram is generated
- quantitative measurement given for viability

Question 27

describe the analysis of measuring apoptosis within a cell population in flow cytometry - method? late vs early apoptosis?

Answer 27

method:
- stains used = propidium iodide, 7-AAD and annexin V conjugated with fluorescin

= PI stains fixed cells - binds and reveals DNA fragments
= 7-AAD stains and enters non-fixed cells with compromised membrane integrity - binds to DNA fragments (induced during late apoptosis)
= annexin V is labelled with fluorescin- binds to phosphatidyl serine, a marker normally on the inner surface of cell membranes but exposed outside in early apoptosis

live, early and late apoptotic cells
- live cells don’t bind to annexin V, 7-AAD or PI
- early apoptotic cells bind annexin with fluorescin as phosphatidyl serine is exposed; no 7-AAD or PI as DNA fragmentation hasn’t occurred yet
- late apoptotic cells bind annexin V, 7-AAD and PI = membrane integrity compromised, DNA fragmented, phosphatidyl serine exposed

Question 28

what is the sub-G0 peak and why is it unreliable?

Answer 28

thought to represent apoptotic cells with DNA fragmentation

unreliable as it doesn’t occur in all cell types, and other assays (annexin V, 7-AAD) are better indicators of apoptosis

Question 29

describe cell sorting/ FACs

Answer 29

FACs allows for the physical separation of cells based on their fluorescence characteristics = sorting based on multiple parameters, at high speed

instead of cells flowing continuously in a stream = nozzle tip is vibrated and cell stream breaks into droplets, each droplet containing a single cell

machine identifies cells that meet a certain criteria - charges the droplets which are deflected by electric fields into collection tubes

sorted cell suspension can be collected in sterile conditions and further experimented on through:
- cell culture
- staining for subpopulation analysis
- microscopy and imaging

Question 30

list some applications for flow cytometry

Answer 30

immunophenotyping of leukaemias and lymphomas
= using antibodies to identity different typed of lymphoma/leukaemia cells based on antigens present

measuring CD4/CD8 cell counts in HIV patients
= monitor numbers and HIV progression, better for treatment

study cell viability, cell cycle and apoptosis

measure cell proliferation or intracellular cytokines