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2 - Cellular Pathology > flow cytometry introductions and applications > Flashcards

flow cytometry introductions and applications Flashcards

1
Q

what is flow cytometry?

A

a technique which uses LIGHT SCATTER and FLUORESCENCE to simultaneously measure several physical characteristics belonging to a SINGLE CELL in SUSPENSION

measuring properties of cells in flow

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2
Q

flow cytometry vs flow sorting

A

Flow Cytometry

  • Measuring properties of cells in flow
  • eg. how many CD4 cells are in this sample of blood

Flow Sorting/Fluorescence-Activated Cell Sorting (FACS)

  • Sorting (separating) cells based on properties measured in flow
  • eg. how many CD4 cells are in this sample of blood, AND give those to me in a separate tube
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3
Q

Flow Sorting/Fluorescence-Activated Cell Sorting (FACS)

A

sorting/separating cells based on properties measured in flow

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4
Q

what can a flow cytometer tell us about a cell?

A
  1. Its Relative Size
  2. Its Relative Granularity/Internal Complexity
  3. Its Relative Fluorescence Intensity
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5
Q

methods of visualisation?

A
  • Fluorescence Microsocopy

- Flow Cytometry

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6
Q

what is CD4?

A

marker on T cells

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7
Q

disadvantages of looking down a microscope vs flow cytometry

A
  • can only look at a limited number of cell in each field when we look down a microscope - means if we are looking for rare cells, we will have to look at thousands of different fields down the microscope
  • not very quantitative as we are looking at cells by eye (we could only look at maybe 20 cells per field, but with the flow cytometer we are looking at thousands of cells every second)
  • the fluorescence is variable – looking by eye and making subjective judgments about the intensity of the fluorescence
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8
Q

Basics of Flow Cytometry

A

fluidics, optics and electronics

  • cells in suspension flow in single-file through an illuminated volume
  • lasers hit the cell, scatter light and emit fluorescence
  • this is collected, filtered and converted to digital values stored on a computer
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9
Q

Fluidics

A
  • Need to have cells in suspension flow as they need to flow in single file
  • Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
  • Sample fluid flows in a central core that does not mix with the sheath fluid - Laminar flow

Introduction of a large volume into a small volume is Hydrodynamic Focusing

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10
Q

Optics - Light Sources

A

Lasers

  • single wavelength of light (a laser line), or more rarely a mixture of wavelengths
  • can provide from milliwatts to watts of light
  • can be inexpensive air-cooled units or expensive water-cooled units
  • provide coherent light (single frequency)
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11
Q

What happens when a laser hits the cell?

A

Light is scattered in 2 directions:

  1. FORWARD SCATTER: scattered in forwards direction, proportional to the size of the cell
  2. SIDE SCATTER: scattered at 90 degree angle and proportional to the granularity/internal complexity of the cell
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12
Q

what is a dot plot?

A

every dot represents an “event”, or a cell

-you can see populations being established, can use this technique to quantify populations in the blood

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13
Q

when you add antibodies with fluorescence, what happens?

A
  • labelled cells with 4 different antibodies with 4 different colours
  • the light is emitted to the cells, and goes through filters and mirrors
  • light is then picked up by photomultiplier tubes, converting light into digital
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14
Q

Electronics

A

Processing of signals from detectors

-Analog-Digital Conversion

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15
Q

explain what is meant by the Stokes Shift?

A

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

(difference between the 2 peaks)

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16
Q

how does fluorescence happen?

A

when the flurochrome is excited by a laser and goes back to its unexcited state, and emits fluorescence at a higher wavelength

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17
Q

most common flurochrome?

A

green

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18
Q

the fluorochromes are excited by?

A

common 488 laser

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19
Q

name some different fluorochromes and their dyes - and their function?

A

FLUOROCHROMES and DYES

Fluorescein isothiocyanate (FITC) GREEN

Phycoerythrin (PE) ORANGE

Peridinin Chlorophyll Protein RED

You would use these flurochromes together to analyse 3 different parameters of the cell. Because they emit different wavelengths, we can detect them all at the same time.

20
Q

for flow cytometry, what do tissues have to be made into?

A

a single cell suspension

21
Q

what are methods of labelling for IMMUNOFLUORESCENCE?

A

DIRECT : Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes

INDIRECT: Unconjugated MoAbs

22
Q

indirect labelling method?

A

-coming in with a second antibody that has a flurophore attached, then it goes through the flow cytometer

23
Q

which labelling method is worse, indirect or direct?

A

indirect

it takes longer and is more complicated, use it if you cannot use a direct antibody.

24
Q

what are the 2 ways of displaying data from a slow cytometer?

A

Histogram and dot plot

25
Q

3 different flurochromes will allow you to distinguish between how many populations?

A

8

26
Q

how is cellular DNA detected?

A

using a fluorescent dye that binds preferentially to DNA

-most common is propidium iodide

27
Q

propidium iodide undergoes what?

A
  • a dramatic increase in fluorescence upon binding to DNA (between DNA base stacks)
  • requires permeabilization of the plasma membrane.
28
Q

how does histogram represent the data?

A

Histogram only looks at 1 parameter
-x- axis is fluorescence intensity
-y –axis is number of cells
Tells us how many cells there are at each fluorescence intensity - the further along the axis, the brighter the cells are

29
Q

how does the PI assay work?

A

PI cannot normally cross the cell membrane, so if it gets through the membrane is assumed to be damaged

cells that are brightly fluorescent with the PI are damaged or dead

30
Q

with the PI assay why do damaged or dead cells appear bright?

A

because the plasma membrane is damaged, and therefore PI can cross

31
Q

what is apoptosis?

A

programmed cell death where the cell goes through a highly regulated process of “dying”

32
Q

characteristics of apoptosis?

A

Characteristics are condensation of the chromatin material

Blebbing of nuclear material

-often accompanied by internucleosomal degradation of DNA giving rise to distinctive ‘ladder’ pattern on DNA gel electrophoresis

33
Q

what is the gold standard for measuring apoptosis?

A

looking for the distinctive ladder on DNA electrophoresis

34
Q

how can we quantitate apoptosis?

A

using flow cytometry

35
Q

necrosis vs apoptosis?

A

N - cell membrane ruptures and releases the cell content and the organelles are not functional

A - the cell breaks into several apoptotic cell bodies, the organelles are still functional

36
Q

3 detection methods for apoptosis?

A

staining with PI dye

Phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)

By staining with 7-aminoactinomycin D (cells not fixed)

37
Q

how can Phosphatidyl serine be used to detect apoptosis?

A

Phosphatidyl serine is normally inside the cell, but when the cell undergoes apoptosis it flips onto the outside of the plasma membrane
-we can exploit this fact by adding the fluorescently labelled Annexin V

38
Q

explain what is meant by sub-G0 apoptotic cells?

A

with the sub g0 peak, people think it is apoptotic cells

  • BUT some people think its debris, and also some cells that undergo apoptosis don’t have this peak, so this statement isn’t entirely accurate
  • add an agent into the cells to initiate apoptosis, and then stain with PI at various times - it can be seen that the G-o peak is increasing
39
Q

A live cell is negative for both PI and Annexin-FITC – why?

A
  • because PI cannot enter the cell because there is no damaged membrane
  • because Annexin-FITC binds to P.S., but in a live cell P.S. is on the inside of the cell so it can’t bind to it
40
Q

In an apoptotic cell, the cells will be positive for Annexin-FITC but negative for PI - why?

A

P.S. will be flipped to the outside, so Annexin-FITC can bind

HOWEVER, PI cannot get in because the plasma membrane is still not damaged

41
Q

when the cells travel further down the apoptotic pathway or they are dead - the cells are positive for both red and green - why?

A

because the PS is on the outside AND the membrane is damaged

42
Q

7-Aminoactinomycin D (7-AAD)

A

Excite it with the 488 laser, and it will emit far out - long emission wavelength

DNA-specific - intercalates in G-C regions

7AAD gives a characteristic 3 populations:

  • live cells are negative, apoptotic cells are dim and dead cells are bright
  • put cell through the sorter and purify them
43
Q

how can caspase pathway be measured?

A

flow cytometry, so we can quantify different parts of the pathway

44
Q

applications of flow cytometry?

A

Immunophenotyping of leukaemias & lymphomas
-they are cells at different stages of the cell life, so they have different antigen profiles

Used to measure CD4/CD8 ratios in HIV patients

Measurement of cell proliferation - use dyes that stain the plasma membrane, and as the cell divides the fluorescence will divide in 2 each time

Detection of MRD

Stem cell enumeration

Measurement of intracellular cytokines

Study of cell cycle, viability & apoptosis

Assessment of transfection efficiency

45
Q

explain what happens in cell sorting?

A

cells are introduced into the nozzle tip, and then leave the nozzle tip

the laser hits the cells as they are flowing in single file, florescence is emitted

the nozzle tip is vibrated so that the cells stay in flow for a certain amount of time, and then the vibrations break them into droplets

machine charges the stream and the drop is pulled via deflection plates into tubes

-stream is charged when there is a cell that satisfies the population

can sort region 1 and 2 at the same time

2 - Cellular Pathology (24 decks)

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